High Mcl-1 Expression Research Articles

Abstract 4652: Combined inhibition of MCL-1 and MEK reduces tumor growth in a triple-negative/inflammatory breast cancer, MEK-resistant xenograft mouse model

Abstract Background: Triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) are highly aggressive subtypes of invasive breast cancer. Our previous work showed that the MEK inhibitor (MEKi) selumetinib (AZD6244) prevents lung metastasis in a xenograft model. However, clinical studies have shown that MEKi’s as single agents have only modest activity against solid tumors. Using a genome-wide synthetic lethal siRNA screening, we identified myeloid cell leukemia-1 (MCL1) as a potential contributor to selumetinib resistance. MCL-1 is an anti-apoptotic protein associated with cell immortalization, transformation, and chemoresistance in numerous cancers. We hypothesized that MCL-1 promotes MEKi resistance in TNBC/IBC and inhibition of MCL-1 in the presence of AZD6244 overcomes this MEKi resistance. Methods & Results: To understand MCL1’s role in MEKi resistance, we established two AZD6244 -resistant TNBC/IBC cell lines: MDA-MB-231-R and SUM149-R. These cells showed increased cell viability, colony formation, migration, cells in G1 phase, CD44+CD24low, mammosphere count, anchorage-independent growth and high MCL-1 expression. MCL-1’s biological role was evaluated by transient and stably knock down (KD) of MCL-1 in the resistant cells which showed decreased cell viability, colony formation, and increased apoptosis. To understand the mechanism mediating apoptosis, we examined the expression of MCL-1’s binding partners in the MCL-1-KD-R cells in the presence of AZD6244. These cells showed a high expression of the PUMA, NOXA, BAK, and BAX in the presence of AZD6244. Further, we treated resistant cells with MEKi AZD6244 and an MCL-1i (AZD5991) that confirmed restoration of MEK sensitivity in both resistant cell lines, resulting in decreased cell viability, colony formation and increased apoptosis. We compared tumor formation capability between SUM149 parental and SUM149 resistant cells by injecting them in nu/nu mice and observed a time-dependent increase (avg. 4-fold) in tumor growth with SUM149-R cells compared to parental cells (P≤0.0001). Moreover, IHC analysis showed high expression of MCL-1 (P≤0.01), Ki67 (ns), CD44 (P≤0.0001), and ALDHA1 (P≤0.0001) in the mammary gland tumor tissues of SUM149-R-injected mice compared to SUM149-P-injected mice. Next, we evaluated the therapeutic effect of AZD6244 in mice harboring SUM149-R - MCL-1-deficient tumors. AZD6244 treatment significantly reduced tumor development in mice harboring SUM149-R - MCL-1-KD tumors compared to control mice (P<0.0001), suggesting that MCL-1 mediates MEK resistance in triple-negative/inflammatory breast cancer. Conclusion: Our results suggest that MCL-1 is important for inducing acquired MEK resistance and combining an MCL-1i with a MEKi could improve treatment outcomes of patients with TNBC or IBC who have developed resistance to single-agent MEKi. Citation Format: Mohd Mughees, Alex Tan, Lakesla Iles, Michael White, Debu Tripathy, Savitri Krishnamurthy, Jian Wang, Wendy A. Woodward, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Combined inhibition of MCL-1 and MEK reduces tumor growth in a triple-negative/inflammatory breast cancer, MEK-resistant xenograft mouse model [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 4652.

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.

Effect of venetoclax plus chemotherapy on treatment-naive acute myeloid leukemia patients with moderate to poor cytogenetic profiles and the combination's influence on the expression of proteins of the anti-apoptoic family

Objective: This was an open-label observational assessment aimed to evaluate whether venetoclax (VEN) plus chemotherapy could enhance the therapeutic benefits for treatment-naive acute myeloid leukemia (AML) patients with adverse cytogenetic profiles. Methods: A total of 38 adult patients (including 11 patients with moderate risk stratification and 27 patients with high risk stratification) who were treated at the Affiliated Hospital of Inner Mongolia Medical University from April 2019 to May 2022 were enrolled in this study. Patients were randomized into two cohorts according to the random number method to receive single intensive chemotherapy (18/38) alone or VEN+intensive chemotherapy (20/38), respectively. The chemotherapy cohort received 2 cycles of induction chemotherapy (idarbicin or daunorubicin plus cytarabine), followed by 6 cycles of consolidation chemotherapy (cytarabine), while the treatment for the VEN + chemotherapy cohort consisted of the same chemotherapy as above plus oral VEN. Heparinized bone marrow samples were obtained from patients at enrollment de novo and post chemotherapy. The expressions of MCL-1 and BCL-2 were detected by Western blot analysis. Results: Patients with VEN+chemotherapy showed an objective response rate (ORR) of 90.0% (18/20), compared with 55.6% (10/18, P=0.012) of the chemotherapy group. Meanwhile, the VEN + chemotherapy cohort gained more benefits in progression-free survival (PFS) and overall survival (OS) than the chemotherapy cohort (mean PFS: 27.1 months versus 17.9 months, P=0.038; mean OS: 32.2 months versus 21.3 months, P=0.004). For patients with moderate risk stratification, there were no differences in the ORR and PFS between the chemotherapy cohort and the VEN + chemotherapy cohort: the ORR was 80.0% (4/5) versus 100% (6/6, P=0.251), and the PFS was 27.9 months versus 32.0 months (P=0.582). Moreover, the ORR was 85.7% (12/14) for the VEN+chemotherapy cohort and 46.2% (6/13) for the chemotherapy cohort in the high risk profile (P=0.029). The PFS of the VEN+chemotherapy cohort was superior to the chemotherapy cohort in the high risk profile (mean PFS: 23.7 months versus 11.1 months, P=0.002). Meanwhile, in the chemotherapy cohort, there were no difference in the PFS between FAB-M5 patients and non-FAB-M5 patients; the mean PFS was 20.0 months versus 15.5 months (P=0.298) for the two groups. Nevertheless, FAB-M5 patients were inferior to non-FAB-M5 patients in PFS in the VEN + chemotherapy arm (mean PFS: 19.6 months versus 30.2 months, P=0.031). The most frequent grade 4 hematological toxicities (therapy related) were leukopenia and thrombopenia. Grade 3/4 hematological adverse events in patients treated with VEN+chemotherapy were not increased compared with those who received chemotherapy. Western blot showed VEN continuously decreased the expression of BCL-2 proteins in both FAB-M5 and non-FAB-M5 patients, but obviously increased the expression of MCL-1 proteins only in FAB-M5 patients. Conclusions: VEN combined with intensive chemotherapy have yielded high ORR and survival advantages for de novo AML patients with poor cytogenetics profiles. The high-expression of MCL-1 may drive resistance to VEN.

Preclinical Evaluation of T-Cell Prolymphocytic Leukemia Demonstrates Heterogeneous BCL2-Family Dependency That May be Effectively Targeted with Small Molecule Inhibitors

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, mature T-cell leukemia with very few treatment options and adverse prognosis. There are currently no approved therapies for patients with relapsed T-PLL, where the median OS is less than 6 months. There are limited reports suggesting clinical activity of the BCL2 inhibitor venetoclax in patients with T-PLL. We aimed to characterize the dependencies of T-PLL on members of the BCL2-family of antiapoptotic proteins using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis (priming) and the relative dependence of a cell on different antiapoptotic proteins. We then sought to experimentally exploit these dependencies with targeted therapies alone, and in combination, designed to inhibit the antiapoptotic proteins towards a therapeutic goal. Cell lines of mature T cell lymphomas/leukemias (TCL/L) including SUPT11 (T-cell leukemia with rearranged TCL1) (n=10) and bone marrow and peripheral blood samples from untreated patients with T-PLL (n=7) were analyzed by BH3 profiling. We also used a relapsed/refractory T-PLL patient sample that were able to engraft and expand in a PDX model. Finally, scRNA/ATAC seq has also been performed in a set of naïve-treated T-PLL patients. In contrast to TCL/L that demonstrated heterogeneity on BCL2 family member dependency, T-PLL samples were primed and consistently exhibited dependency on BCL2/BCL-xL and MCL1. Integration of scRNA/ATAC seq confirmed high expression of BCL2 and MCL1 in T-PLL cells from four patients supporting the premise that BCL2 and MCL1 could potentially be targeted for therapeutic benefit. Therefore, SUPT11 cells (used as a model of T-PLL) were exposed to venetoclax (25 and 50nM; BCL-2inh), fadraciclib (25 and 50nM; CDK2/9inh - indirect inhibitor of MCL-1 as it decreases mRNAs with high decay rates including MCL1 and MYC) or a combination of venetoclax and fadraciclib. We demonstrate that exposure to fadraciclib caused a rapid decline in the levels of phosphorylation on Serine 2 of RNA polymerase II C-terminal domain, as well as decline in the levels of MCL1 within 4 hours and completely depleted these proteins by 8-12 h of exposure. We also show that there was a synergistic increase both in mitochondrial dysfunction as measured by cytochrome C release by 18h and in cell death in cells exposed to the combination at 24h. These results suggest that combining venetoclax and fadraciclib might represent a potential therapeutic approach for T-PLL. In conclusion, preclinical evaluation of a T-PLL cell line and primary patient samples demonstrate BCL2-family dependency that can be effectively targeted with small molecule inhibitors of BCL2 and CDK2/9. In vivo studies in PDX models of T-PLL are underway to form the basis for clinical trials to study these combinations.

Correlation of Flow Cytometric BCL2 and MCL1 Expression with Cytogenetic Characteristics and Outcome in Multiple Myeloma

Introduction: Clonal plasma cells mediate apoptosis resistance through increased expression of anti-apoptotic proteins which increase survival and mediate resistance to intrinsic mitochondrial pathway. The key anti-apoptotic members are BCL-2, MCL-1, BCL-XL, BCL-B and BCL-W. High BCL2 expression or t(11;14) is shown to be predictive of response to Venetoclax in Multiple Myeloma (MM). Venetoclax may only be effective in a subset of patients, namely those who have relatively high BCL2 and low MCL1. Resistance to Venetoclax can be mediated by mutation in BCL2 or gain in copy number of MCL1, which maps to 1q21. The relationship between cytogenetic abnormalities and expression of BCL2 and MCL1 is not clear except for an occasional study which has shown MM with CCDN1 translocation has high BCL2 and low MCL1 expression and MM with MMSET and MAF translocation has high MCL1 and low BCL2 expression. Also, previous studies have used either immunohistochemistry or real time quantitative PCR for studying BCL2 and MCL1 expression, both of which are met with challenges in interpretation. We aimed to correlate the intracellular expression of BCL2 and MCL1 by flow-cytometry (FCM) with cytogenetic characteristics and outcome. Methodology: FCM was performed in bone-marrow of 143 consecutive MM cases with ≥10% plasma cells (PCs) and 20 bone marrow samples with non-clonal PCs by lyse-stain-wash method followed by intracellular staining of BCL2 (Alexa Fluor 647 conjugated anti-human BCL2 antibody) and MCL-1 (PE-conjugated anti-human MCL1 antibody). The sequential gating strategy used in the analysis of intracellular BCL2 and MCL1 expression in PCs and T lymphocytes is shown in Figure 1. The percentage of PCs positive for BCL2 and MCL1 compared to the tube without these antibodies [Fluorescence Minus One (FMO) cutoff threshold], the mean fluorescence intensity (MFI) expression of BCL2 and MCL1 in PCs and T cells, and the ratio of expression of BCL2 and MCL1 on PCs and T cells (BCL2 PC/T, MCL1 PC/T), MFI BCL-2 PC/MFI MCL1 PC were studied and further correlated with 5-7 panel FISH cytogenetics by using Kruskal Wallis test or Mann-Whitney U test. The overall survival/OS and progression-free survival/PFS was calculated using Kaplan-Meir Survival Curve analysis. Result and discussion: During the study, FCM was performed in 143 MM patients (103 Newly Diagnosed MM and 40 Relapsed Refractory MM). The cytogenetic findings includes IGH:: FGFR3 (n=29), IGH:: CCND1 (n=8), IGH:: MAF (n=3), gain1q21 (n=76), 1q21-amplification (n=30), 1p32-deletion (n=17) and TP53-deletion (n=21). A median of 38,779 PCs was acquired by FCM. The BCL2 and MCL1 expression was seen in 4-100% (median-96.9%) and 0-100% (median-78.8%) of clonal PCs, respectively. The median BCL2 MFI and MCL1 MFI were 43,289 and 6,481 respectively. The median BCL2 PC/T, MCL1 PC/T and BCL2 PC/MCL1 PC in clonal PCs were 1.90, 4.35, and 07.1, respectively, whereas in non-clonal PCs they were 0.87, 3.02 and 3.21, respectively.BCL2 PC/T, MCL1 PC/T and BCL2 PC/MCL1 P were significantly higher in clonal PCs than non-clonal PCs, with a p-value of 0.002, 0.001, and 0.009, respectively. The median MCL1 MFI was significantly lower (3,825 vs. 6,559; p=0.020) and median BCL2 PC/MCL-1 PC (12 vs 6.7; p=0.012) was significantly higher in IGH:: CCND1 compared to others; while an opposite pattern was seen in gain1q21 (7,420 vs 5,525; p=0.032 and 5.5 vs 8.6; p=0.046). The dim expression (<25-percentile) of BCL2 and MCL1 was noted in 25% (n=36) MM cases. Survival analysis did not yield a significant difference in OS or PFS between patients with dim, moderate or strong expression of BCL2 or MCL1. Conclusions: Our study demonstrates an objective assessment of BCL2 and MCL1 expression by FCM. BCL2 PC/T,MCL1 PC/T and BCL2 PC /MCL1 PC were significantly higher in myeloma cells compared to non-clonal PCs. Low MCL1 expression or high BCL2/MCL1 ratio was associated with IGH:: CCND1; while strong MCL1 and low BCL2/MCL1 ratio was most frequent in 1q21 amplification. We identified dim-expression (< 25-percentile) of BCL2 or MCL1 in 25% of cases of MM, where the benefit of anti-BCL2/MCL1 targeted therapy needs to be studied. Routine testing of BCL2 and MCL1 expression by FCM may be beneficial before starting Venetoclax/similar targeted therapy.

Increased MCL1 dependency leads to new applications of BH3-mimetics in drug-resistant neuroblastoma

BackgroundNeuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells.MethodsWe utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance.ResultsIn a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells.ConclusionsThese data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.

High-expression of the innate-immune related gene UNC93B1 predicts inferior outcomes in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with dismal prognosis. Identification of better biomarkers remained a priority to improve established stratification and guide therapeutic decisions. Therefore, we extracted the RNA sequence data and clinical characteristics of AML from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression database (GTEx) to identify the key factors for prognosis. We found UNC93B1 was highly expressed in AML patients and significantly linked to poor clinical features (p < 0.05). We further validated the high expression of UNC93B1 in another independent AML cohort from GEO datasets (p < 0.001) and performed quantitative PCR of patient samples to confirm the overexpression of UNC93B1 in AML (p < 0.005). Moreover, we discovered high level of UNC93B1 was an independent prognostic factor for poorer outcome both in univariate analysis and multivariate regression (p < 0.001). Then we built a nomogram model based on UNC93B1 expression, age, FAB subtype and cytogenetic risk, the concordance index of which for predicting overall survival was 0.729 (p < 0.001). Time-dependent ROC analysis for predicting survival outcome at different time points by UNC93B1 showed the cumulative 2-year survival rate was 43.7%, and 5-year survival rate was 21.9%. The differentially expressed genes (DEGs) between two groups divided by UNC93B1 expression level were enriched in innate immune signaling and metabolic process pathway. Protein-protein interaction (PPI) network indicated four hub genes (S100A9, CCR1, MRC1 and CD1C) interacted with UNC93B1, three of which were also significantly linked to inferior outcome. Furthermore, we discovered high UNC93B1 tended to be infiltrated by innate immune cells, including Macrophages, Dendritic cells, Neutrophils, Eosinophils, and NK CD56dim cells. We also found UNC93B1 had a significantly positive correlation with CD14, CD68 and almost all Toll-like receptors. Finally, we revealed negatively correlated expression of UNC93B1 and BCL2 in AML and conjectured that high-UNC93B1 monocytic AML is more resistant to venetoclax. And we found high MCL-1 expression compensated for BCL-2 loss, thus, we proposed MCL-1 inhibitor might overcome the resistance of venetoclax in AML. Altogether, our findings demonstrated the utility of UNC93B1 as a powerful poor prognostic predictor and alternative therapeutic target.

Unraveling the Heterogeneity of Multiple Myeloma Cells By Single-Cell RNA Sequencing Analysis

Multiple myeloma (MM) is a malignancy of clonal plasma cells with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire) and extensive genome or transcriptome heterogeneity. Even though many new treatment modalities have been developed and the prognosis of patients has improved significantly, most cases of MM remain incurable. Recent evidence suggests that non-genetic cell plasticity and changes in cell state underlie the therapy resistance and tumor relapse, but the mechanisms remain largely unknown. Here, we have attempted to understand comprehensive architecture of the entire tumor cell populations using repertoire clonality as fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions based on the known surface antigens of MM and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were also present in some minor fractions other than the main fraction (Lin-/CD19-/CD38++/CD138+) as the stage progressed. Importantly, cells with clonal repertoire were detected in CD138 negative fraction (Lin-/CD19-/CD38++/CD138-) in most samples (Monoclonal gammopathy of undetermined significance (MGUS) / Smoldering multiple myeloma (SMM) = 4.15±2.28%, Primary MM = 6.78±3.59%, Relapse MM = 7.8±5.82% [mean±95%Cl], n=24). The CD138 positive and negative cells were also morphologically different, suggesting that they were composed by cells of distinct characteristics. Indeed, comparison of the transcriptome data revealed 296 differentially expressed genes (DEGs) between these fractions (250 up-regulated and 46 down-regulated in CD138 negative fractions). Of interest, several pathways related to H3K4 methylation were positively enriched in CD138 negative fraction (p <0.001), which included KMT2A, KMT2B, KMT2C, KMT2D, and ASH1L, suggesting that differential methylation of histone H4 accounts for the heterogeneity of MM. In order to achieve a high-resolution and comprehensive evaluation of the entire tumor cell populations, we performed single-cell RNA sequencing (scRNA-seq) and single-cell VDJ targeted sequencing (scVDJ-seq) concurrently on Lin-/CD38++ sorted cells of 8 bone marrow samples from 7 patients including pre- and post-treatment status samples. This method allowed us to define normal cells and MM cells at the single-cell level by clonal repertoire sequence independent of transcriptome characteristics. We divided 18,031 MM cells into 23 clusters after integration. We first defined CD138 positive and negative MM gene signature using bulk RNA-seq data and then evaluated the scRNA-seq data. Notably, the CD138 axis appeared to clearly subdivide main MM populations into those with CD138 positive and negative MM gene signature. We also found that a part of clusters, which showed CD138 negative MM gene signature, tended to remain at high proportions after treatment. These populations were characterized by low expression levels of MHC class-I components and TNFRSF17/BCMA as well as high expression of MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance, and were present in all samples. H3K4 methylation related genes were also up-regulated in these populations. Furthermore, we found several clusters with unique and interesting characteristics independent of the CD138 axis. Trajectory and velocity analysis suggested that all cell populations transit to each other. Taken together, our results establish the heterogeneity of MM cells by the CD138 axis, which may be characterized by epigenetic plasticity. They also highlight CD138 negative population as a potential cause of treatment resistance and relapse. By using information of repertoire, we have achieved a comprehensive and accurate single-cell analysis of MM cell diversity that was not limited by cell surface antigens or transcriptome characteristics. We are currently analyzing near minimal residual disease (MRD) level samples using the same scRNA-seq methods and the functional and epigenetic properties of CD138 positive and negative MM cells.

All-trans retinoic acid enhanced the antileukemic efficacy of ABT-199 in acute myeloid leukemia by downregulating the expression of S100A8

Acute myeloid leukemia (AML) is prone to relapse. Targeted therapy with a specific inhibitor of the anti-apoptotic protein Bcl-2 ABT-199 is an effective method for relapsed and refractory patients, but drug resistance is likely, which is primarily related to high Mcl-1 and S100A8 expression. All-trans retinoic acid (ATRA) can inhibit Bcl-2 and Mcl-1 expression. The study purpose was to determine whether ATRA can enhance the antileukemia effect of ABT-199 on AML cells. Our data showed that ATRA combined with ABT-199 exerts a synergistic antileukemic effect by inducing apoptosis and cell cycle arrest in AML. In vivo, combination therapy prolonged the survival of AML xenograft mice. The possible mechanism involves promoting apoptosis through downregulation of S100A8 expression by inhibiting the PI3K/AKT signaling pathway. This study provides a potential treatment strategy and theoretical support for overcoming the clinical ABT-199 resistance problem in AML patients.

Increased apoptotic sensitivity of glioblastoma enables therapeutic targeting by BH3-mimetics

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.

Abstract P5-13-06: Exploratory biomarker analysis in VERONICA, a phase 2 study of venetoclax + fulvestrant versus fulvestrant in patients with estrogen receptor (ER)-positive HER2-negative metastatic breast cancer (mBC)

Abstract BACKGROUND: Venetoclax (VEN) is a potent and selective inhibitor of the anti-apoptotic protein, BCL2. Preclinical studies have implicated the BCL2 family members, BCLXL and MCL1, in VEN resistance, and clinical studies in hematological malignancies have demonstrated subgroups with high ratios of BCL2/BCLXL and BCL2/MCL1 have the greatest VEN antitumor activity. The randomized phase 2 VERONICA study (NCT03584009) evaluated VEN in combination with fulvestrant (F) vs F alone in ER-positive, HER2-negative MBC pts who experienced disease recurrence/progression during or after a CDK4/6 inhibitor. Previously reported results from VERONICA (Lindeman et al. ASCO 2021) did not show an improved clinical benefit rate or progression-free survival (PFS) with VEN+F vs F alone. Here we present exploratory biomarker analyses of the expression of BCL2 family members and genomic alterations in circulating tumor DNA (ctDNA) and association with clinical outcomes from VEN+F vs F. METHODS: Tumor specimens were obtained during screening from 103 patients enrolled in the study, and expression levels of BCL2, BCLXL and MCL1 were analyzed by IHC. Baseline plasma-derived ctDNA was evaluated using the FoundationOne® Liquid assay. Expression of BCL2, BCLXL, MCL1 and mutations in ctDNA were correlated with PFS from VEN+F vs F based on the primary analysis (cutoff: Aug 5, 2020). RESULTS: In the overall population, protein levels of BCL2, BCLXL and MCL1 were similar between the VEN+F vs F arms. Patients whose tumors were BCL2 3+ trended towards having the greatest difference in median (m) PFS (3.9 months [mo] in VEN+F vs 1.7 mo in F; hazard ratio [HR] 0.38 [95% CI 0.09, 1.62]) albeit in a small sample size (n=13). Similarly, subgroup analysis suggested a trend for increasing mPFS and improved HR in VEN+F vs F alone in patients with the lowest BCLXL expression. mPFS in patients with a BCL2/BCLXL ratio ≥1 was 3.7 mo for VEN+F vs 1.8 mo for F (HR 0.67 [95% CI 0.3-1.49]) whereas patients with a BCL2/BCLXL ratio &amp;lt;1 had no difference in mPFS between the arms (2.0 mo in both arms, HR 1.21 [95% CI 0.7-2.1]). In the ctDNA-evaluable population, ESR1 (42.6%), TP53 (41.5%) and PIK3CA (35%) were the most prevalent genomic alterations observed and well-controlled between arms. PFS was similar between the ESR1 wildtype (wt) and mutant (mut) subgroups. TP53 mut status was a poor prognostic factor in both treatment arms. The PIK3CA wt subgroup had increased mPFS with VEN+F vs F alone (HR 0.66 [95% 0.38-1.17]) compared to PIK3CA mut (HR 1.59 [95% 0.74-3.34]). Patients with PIK3CA wt and BCL2 high tumors had the largest difference in mPFS between VEN+F (3.7 mo) vs F alone (1.9 mo) (HR 0.58 [95%CI 0.28-1.19]), compared to PIK3CA wt-BCL2 low tumors (2.4 vs 1.9 mo; HR 0.67 [95%CI 0.26-1.72]). CONCLUSION: Our data suggest that a high ratio of BCL2 to BCLXL conferred a trend towards a greater benefit to VEN+F compared to F alone, consistent with other clinical studies evaluating VEN. These analyses highlight the need to profile BCL2 and its family members to identify the VEN-sensitive subgroups, especially in indications where high expression of BCLXL or MCL1 may be observed. The ctDNA profile of VERONICA patients indicate a heavily pretreated patient population. The benefit observed in PIK3CA wt patients from VEN+F suggest increased dependence on BCL2 in this subgroup, while PIK3CA mut tumors likely rely on PI3K/AKT/mTOR survival programs or other BCL2 family members to evade apoptosis. Exploratory biomarker analyses are ongoing to further understand the VERONICA patient population. Citation Format: Geoffrey J. Lindeman, Tharu M. Fernando, Rebecca Bowen, Ching-Wei Chang, Rupal Desai, Kushagra Gupta, Aulde Fléchais, Timothy R. Wilson, Aditya Bardia. Exploratory biomarker analysis in VERONICA, a phase 2 study of venetoclax + fulvestrant versus fulvestrant in patients with estrogen receptor (ER)-positive HER2-negative metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-06.

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

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

Association between BCL2, BCL2 E17, MCL1 and BAX Protein Expression, Bone Marrow Microenvironment Histological Features, Clinical Presentation, Therapeutic Outcome, and the Overall Survival in Newly Diagnosed Multiple Myeloma

Association between BCL2, BCL2 E17, MCL1 and BAX Protein Expression, Bone Marrow Microenvironment Histological Features, Clinical Presentation, Therapeutic Outcome, and the Overall Survival in Newly Diagnosed Multiple Myeloma

Leukemic Stem Cells of Monocytic AMLs Are Not-Resistant to BCL-2 Inhibition

Treatment with Hypomethylating agents (HMA) such as 5-Azazytidine (AZA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows results superior to treatment with AZA alone (DiNardo et al., 2020, NEJM). However upfront resistance and relapse following initial response remain major obstacles.It has recently been proposed that monocytic differentiation predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). This appears to be due to increased expression of other anti-apoptotic proteins such as MCL-1 in monocytic AMLs, which conveys resistance to AZA/VEN therapy, as survival of leukemic cells in these patients is no longer dependent on BCL-2. However, an independent study found no impaired outcome in patients with monocytic AMLs treated with HMA/VEN (Maiti et al., 2020, Blood, ASH abstract).Here, we show that monocytic AML cell lines and bulk cells of monocytic primary AML cells are indeed intrinsically resistant to AZA/VEN treatment. However, in a collective of 30 patients treated with HMA/VEN at Heidelberg University Medical Center between 2018 and 2020, monocytic differentiation assessed by flow cytometry was not an independent risk factor for refractory disease. We hypothesized that the conflicting data may be caused by intra-patient heterogeneity of AZA/VEN sensivitity and assessed killing efficiency in various immunophenotypic subpopulations of 12 primary AML patient samples in vitro.The CD64 +CD11b +, differentiated blast population made up >50% of leukemic cells in monocytic and <20% in primitive samples and showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain leukemic stem cells (LSC).In contrast, we found immature CD64 -CD11b - GPR56 + LSC to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive types of primary AMLs. As expected, LSCs from either monocytic or primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential for the effect of AML therapy.Next, we investigated expression of BCL-2, MCL-1 and BCL-xL in the same primary patient samples and observed high MCL-1 expression in monocytic AML samples. However, MCL-1 expression was restricted to the CD64 +CD11b + population whereas in the LSC sub-populations robust expression of BCL-2 but low levels of MCL-1 and BCL-xL were detected, independent of whether monocytic or primitive AMLs were analyzed.To further validate the sensitivity of LSCs of monocytic AML to BCL-2-I, we established a platform combining BH-3 profiling with multi-color flow cytometry, allowing for single cell assessment of cellular dependencies on independent apoptotic pathways. We found that LSCs of both AML types show high VEN/BAD but low MS-1 induced apoptosis, functionally confirming the expression patterns of BCL-2 and MCL-1. As LSCs are rare in monocytic samples, investigation of samples in bulk are dominated by MCL-1 expressing and resistant non-LSCs, explaining the overall higher MCL-1 expression/survival of monocytic compared to immature AML cells. However, our data uncovers sensitivity of LSCs to AZA/VEN independent of overall monocytic or primitive sample classification and provide a mechanistic explanation for the clinical data of Maiti et al. and our Heidelberg AML collective, which found no increased resistance of monocytic AMLs to AZA/VEN treatment. DisclosuresUnglaub: JazzPharma: Consultancy, Other: travel costs/ conference fee; Novartis: Consultancy, Other: travel costs/ conference fee. Schlenk: Abbvie: Honoraria; Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding.

The Impact of Chlorambucil and Valproic Acid on Cell Viability, Apoptosis and Expression of p21, HDM2, BCL2 and MCL1 Genes in Chronic Lymphocytic Leukemia.

Malignant cells in chronic lymphocytic leukemia (CLL) show resistance to apoptosis, as well as to chemotherapy, which are related to deletions or mutations of TP53, high expression of MCL1 and BCL2 genes and other abnormalities. Thus, the main goal of the present study was to assess the impact of chlorambucil (CLB) combined with valproic acid (VPA), a known antiepileptic drug and histone deacetylation inhibitor, on apoptosis of the cells isolated from 17 patients with CLL. After incubation with CLB (17.5 µM) and VPA (0.5 mM), percentage of apoptosis, as well as expression of two TP53 target genes (p21 and HDM2) and two genes from Bcl-2 family (BCL2 and MCL1), were tested. As a result, an increased percentage of apoptosis was observed for CLL cells treated with CLB and VPA, and with CLB alone. Under the treatment with the drug combination, the expression of p21 gene was visibly higher than under the treatment with CLB alone. At the same time, the cultures under CLB treatment showed visibly higher expression of BCL2 than the cultures with VPA alone. Thus, the present study strongly suggests further investigations on the CLB and VPA combination in CLL treatment.

Promotive effects of HOXA10 antisense RNA on the stemness of oral squamous cell carcinoma stem cells through a microRNA-29a/MCL-1/phosphatidyl inositol 3-kinase/protein kinase B axis

ObjectiveThe aim of this study was to investigate the effects of long non-coding RNA (lncRNA) HOXA10 antisense RNA (HOXA10-AS) on the properties of oral squamous cell carcinoma (OSCC) stem cells and the molecular mechanism. DesignTumor and the paracancerous tissues were collected from 83 patients with OSCC. OSCC stem cells were extracted from a human OSCC cell line Tca8113. Silencing of HOXA10-AS was introduced in stem cells and then the malignant behaviors of cells were determined. The target transcripts of HOXA10-AS were predicted using integrated bioinformatics analyses. The interactions among HOXA10-AS, microRNA (miR)-29a and MCL-1 were validated, and their functions in stem cell behaviors in vivo and in vitro were explored. ResultsHOXA10-AS and MCL-1 were highly expressed while miR-29a was poorly expressed in the collected tumor tissues and the extracted OSCC stem cells. High expression of HOXA10-AS and MCL-1, while poor expression of miR-29a was relevant to poor prognosis in patients. Silencing of HOXA10-AS suppressed proliferation and tumor sphere formation ability of stem cells, and it reduced growth and metastasis of tumors in animals. HOXA10-AS served as a sponge for miR-29a and upregulated MCL-1 mRNA expression. Inhibition of miR-29a promoted, while silencing of MCL-1 suppressed the malignant behaviors of OSCC stem cells. In addition, HOXA-10-AS and MCL-1 were found to activate the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. ConclusionThis study evidenced that HOXA10-AS enhances the stem cell property of OSCC stem cells through the miR-29a/MCL-1/PI3K/AKT axis.

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.

Combination of Homoharringtonine with Venetoclax and Azacitidine Excerts Better Treatment Response in Relapsed /Refractory Acute Myeloid Leukemia

Overexpression of Bcl-2 is known to be an important factor for drug resistance in relapsed /refractory acute myeloid leukemia (R/R-AML). Combination of Bcl-2 inhibitor venetoclax with hypomethylating agents (HMA) has shown promising killing effect in elder patients with newly diagnosed AML, also worked in R/R-AML but with low rate of complete response (CR). High expression of Bcl-XL and/or Mcl-1 is the main reason for resistance to venetoclax. As an inhibitor of both Bcl-XL and Mcl-1, homoharringtonine (HHT) has presented a synegetic effect with venetoclax in inhibition of Mcl-1 and killing of lymphama. In this study, we analyzed the treatment response and safety of combination of HHT (1mg/m2 d1-7) with venetoclax (400mg d1-14) and azacitidine (75mg/m2 d1-7) (HVA) in R/R-AML. From 6, 2019 to 5, 2020, in Nanfang Hopsital, 22 patients with RR-AML were treated with HVA (HVA group), with a median age of 39(16-62) years, previous chemotherapy of 3(1-7) cycles. 15 (68.2%) relapsed within one year after allogenetic hematopoietic stem cell transplantation (allo-HSCT) and 12 (54.5%) previously exposed to HMA treatment. Meanwhile, 7 patients with RR-AML were treated with venetoclax combined with HMA (control group), with a median of 42(31-74) years, previous chemotherapy of 3(1-6) cycles. 2 (28.6%) relapsed after allo-HSCT and 5 (71.4%) had HMA exposure. In HVA group, totally 11/17(64.7%) patients acquired treatment response, and 7/15(46.7%) obtained CR/CRi, of whom 6 presented minimal residual disease (MRD) negative. While in control group, only 2/7(28.6%) acquired treatment response and 1 obtained CR. Subgroup analysis in HVA group showed allover response rate (ORR) was 9/13(69.2%) and CR rate was 6/11(54.5%) (all with MRD negative) in the patients with allo-HSCT. And in the patients without allo-HSCT ORR was 2/4(50.0%) and CR rate was 1/4. With a median follow-up of 6(1-7) months, one patient was bridged to allo-HSCT, 2 out of 7 patients with CR relapsed. All patients were well tolorated to both treatment regimens, showing as a median neutropenia of 9.5(5-14) days, 5 patients with Febrile neutropenia and 4 with pulmonary infection. In conclusion, combination of HHT with venetoclax and azacitidine (HVA) excerts better treatment response in R/R-AML, especially in those with allo-HSCT, and shows well tolorated. A multi-center prospective clinical trial (NCT04424147) has been initiated. Key words: HVA regimen, relapsed /refractory acute myeloid leukemia, treatment response, safety Disclosures No relevant conflicts of interest to declare.

MCL1 inhibition is effective against a subset of small-cell lung cancer with high MCL1 and low BCL-XL expression

There have been few advances in the treatment of small-cell lung cancer (SCLC) because of the lack of targets. MCL1, a member of the anti-apoptotic BCL-2 family, may be a treatment target in several cancers, including SCLC. However, whether the expression profile of the anti-apoptotic BCL-2 family affects MCL1 inhibition strategy is unknown. A tissue microarray (TMA) was created from consecutive patients who were diagnosed with SCLC and had previously undergone surgery at Kyoto University Hospital (Kyoto, Japan) between 2001 and 2017. We used S63845, a MCL1 inhibitor, to assess the cytotoxic capacity in SCLC cell lines including a patient-derived cell line in vitro and in vivo. The combination of S63845 with navitoclax, a double BCL-XL/BCL-2 inhibitor, was also employed to examine the comprehensive inhibition of the anti-apoptotic BCL-2 family. Immunohistochemistry of a TMA from patients with surgically resected SCLC demonstrated high MCL1 expression with low BCL-XL and BCL-2 to be the most common expression profile. S63845 was effective in high MCL1- and low BCL-XL-expressing SCLC cell lines. S63845 induced BAK-dependent apoptosis in vitro, and the anti-tumor efficacy was confirmed in an in vivo model. Although knockdown of BCL-XL and BCL-2 improved the cytotoxic activity of S63845 and its combination with navitoclax increased the anti-tumor cytotoxicity, the therapeutic range of S63845 with navitoclax was narrow in in vivo studies. Our study suggests MCL1 inhibition therapy be applied for high MCL1- and low BCL-XL-expressing SCLC patients.

Venetoclax: the first anti-myeloma agent with a reliable biomarker.

Venetoclax: the first anti-myeloma agent with a reliable biomarker.