Apoptosis In Chronic Lymphocytic Leukemia Research Articles

MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia.

Although clinical outcomes in chronic lymphocytic leukemia (CLL) have greatly improved with several approved small molecular inhibitors, acquired resistance does occur, leading to disease progression and eventual death. Thus, the effort to explore novel inhibitors and combination therapeutic regimens is needed. The inhibition of MDM2-p53 interaction to restore p53 function has been regarded as a potential strategy for treating different cancers. We investigated the effects of novel MDM2 inhibitor APG-115 in CLL. We found that APG-115 treatment upregulated the expression of p53, MDM2, and p21 at the mRNA and protein level. APG-115 inhibited cell proliferation, induced apoptosis, and arrested the cell cycle at G0/G1 stage. Moreover, APG-115 inhibited the expression of BCL-2, BCL-xL, and MCL-1, and suppressed the activation of AKT and ERK signaling pathways. APG-115 combined with the BCL2 inhibitor, ABT-199 (venetoclax), led to further inhibition of the expression of BCL-2 family anti-apoptotic proteins and consequently enhanced cell death. Collectively, this study demonstrates that APG-115 activates p53 and thus inhibits multiple pro-survival mechanisms, which provides a rational explanation for APG-115 efficiency in inducing cell apoptosis in CLL. The synergistic effect of APG-115 with ABT-199 suggested a potential combination application in CLL therapy.

Metabolic profiling of CD19+ cells in chronic lymphocytic leukemia by single-cell mass spectrometry imaging

Background and aimsModern mass spectrometry imaging (MSI) enables single cells’ metabolism exploration. Aims of this study were development of the single-cell MSI of human CD19+ lymphocytes and metabolic profiling of chronic lymphocytic leukemia (CLL). Materials and methodsBlood donor (BD) samples were used for the optimization of CD19+ lymphocyte isolation and single-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) MSI. Independent set of 200 CD19+ lymphocytes coming from 5 CLL patients and 5 BD was used for the CD19+ lymphocytes classification assessment and the untargeted metabolic profiling. CLL vs BD lymphocyte classification was performed using partial least squares-discriminant analysis (PLS-DA) using normalized single-cell mass spectra recorded in 300–600 and 600–950 Da ranges was applied. ResultsAccuracy assessed by 10-fold cross-validation of CD19+ lymphocyte PLS-DA classification reached >90.0 %. Volcano plots showed 106 significantly altered m/z signals in CLL of which 9 were tentatively annotated. Among tentatively annotated m/z signals formaldehyde and glutathione metabolites and tetrahydrofolate stand out. ConclusionA method for single-cell MALDI TOF MSI of CD19+ lymphocytes was successfully developed. The method confirmed the significance of oxidative stress and single-carbon metabolism, pyruvate and fatty acid metabolism and apoptosis in CLL and it provided metabolic candidates for diagnostic applications.

Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter’s syndrome

Richter’s syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

A novel potent class I HDAC inhibitor reverses the STAT4/p66Shc apoptotic defect in B cells from chronic lymphocytic leukemia patients

Chronic Lymphocytic Leukemia (CLL) patients have a defective expression of the proapoptotic protein p66Shc and of its transcriptional factor STAT4, which evoke molecular abnormalities, impairing apoptosis and worsening disease prognosis and severity. p66Shc expression is epigenetically controlled and transcriptionally modulated by STAT4; epigenetic modifiers are deregulated in CLL cells and specific histone deacetylases (HDACs) like HDAC1, are overexpressed. Reactivation of STAT4/p66Shc expression may represent an attractive and challenging strategy to reverse CLL apoptosis defects. New selective class I HDAC inhibitors (HDACis, 6a-g) were developed with increased potency over existing agents and preferentially interfering with the CLL-relevant isoform HDAC1, to unveil the role of class I HDACs in the upregulation of STAT4 expression, which upregulates p66Shc expression and hence normalizes CLL cell apoptosis. 6c (chlopynostat) was identified as a potent HDAC1i with a superior profile over entinostat. 6c induces marked apoptosis of CLL cells compared with SAHA, which was associated with an upregulation of STAT4/p66Shc protein expression. The role of HDAC1, but not HDAC3, in the epigenetic upregulation of STAT4/p66Shc was demonstrated for the first time in CLL cells and was validated in siRNA-induced HDAC1/HDAC3 knock-down EBV-B cells. To sum up, HDAC1 inhibition is necessary to reactivate STAT4/p66Shc expression in patients with CLL. 6c is one of the most potent HDAC1is known to date and represents a novel pharmacological tool for reversing the impairment of the STAT4/p66Shc apoptotic machinery.

Abstract 2102: Understanding the mechanism of action of rigosertib in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma through single cellanalysis

Abstract The allosteric inhibitor rigosertib (an experimental therapeutic) was previously identified as an effective inducer of G2M arrest and apoptosis in squamous cell carcinoma (SCC) keratinocytes isolated from patients with the rare genetic disease recessive dystrophic epidermolysis bullosa (RDEB), both in vitro and in vivo. Early clinical trial data treating RDEB-associated SCC with rigosertib has shown remarkable efficacy in a sub-set of patients and understanding the mechanism of action of rigosertib in this lethal cancer would provide rationale for patient stratification. Multiple mechanisms of action were previously attributed to rigosertib, including polo-like kinase-1 (PLK1), PI3-kinase, activated RAS and microtubule polymerization inhibition. Our published data comparing PLK1 siRNA with PI3-Kinase inhibitors, MAPK inhibitors and microtubule disruption favored PLK1 as a mechanism of action without definitive conclusion. We therefore sought to determine the mechanism of action using transcriptomic profiling. Flow cytometry experiments identified three main categories of time to increase G2M in nine separate RDEB SCC keratinocyte populations in culture ranging from 1 to 6 hours after exposure to 1uM rigosertib. Bulk-RNA sequencing of representative populations from time to G2M groupings failed to identify consistent transcriptional change after exposure to rigosertib. However, single cell sequencing was able to identify increases in sub-populations of cells associated with G2M as well as transient emergence of unique populations that were specific to individual patient populations after exposure to rigosertib in culture. Of note a transient population of RDEB SCC keratinocytes only present after 1 hour of exposure to rigosertib was characterized by increased expression of the potassium regulator KCNRG which has previously been associated with apoptosis in chronic lymphocytic leukemia. Ongoing work is determining the role this gene may play in the mechanism of apoptosis induction in RDEB SCC keratinocytes after exposure to rigosertib. Citation Format: Andrew P. South, Suhao Han, Paolo Fortina. Understanding the mechanism of action of rigosertib in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma through single cellanalysis [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 2102.

Therapeutic targeting of apoptosis in chronic lymphocytic leukemia

Therapeutic targeting of apoptosis with small molecule B-cell lymphoma 2 (BCL-2) inhibition with venetoclax is highly efficacious in CLL, leading to sustained deep responses, particularly among patients with treatment-naïve disease with favorable prognostic markers. Patients with unfavorable genetic characteristics such as TP53 aberration and unmutated IGHV may also derive durable benefits, but their remission duration after time-limited venetoclax-containing combination therapy is shorter, particularly in patients with relapsed/refractory disease. Emerging data indicate that the context of disease progression after initial treatment with venetoclax may define the success of re-treatment with venetoclax. Specifically, continuous venetoclax exposure may select for resistant disease due to genetic mechanisms such as BCL2 mutations and functional resistance mechanisms such as hyperphosphorylation of BCL-2 family proteins, which decrease the affinity of venetoclax binding to the target or lead to increased MCL-1 dependence and concomitant decrease in BCL-2 dependence. These patients may be best served by switching to a different class of targeted agents at the time of progression. In contrast, relapsed CLL that arises while being off therapy after a period of time-limited venetoclax-based regimens maintains sensitivity to re-treatment with venetoclax for the majority of patients. Novel strategies related to therapeutic targeting of apoptosis include next-generation BCL-2 inhibitors with improved potency and pharmacokinetic profiles, direct targeting of anti-apoptotic BH3 family proteins beyond BCL-2 such as MCL-1, and indirect targeting of MCL-1 through mechanisms such as small molecule cyclin-dependent kinase 9 inhibitors.

The Gene Expression Landscape of BTK Inhibitor Treatment in Chronic Lymphocytic Leukemia Exposed By Shallow Depth RNA Sequencing

Efforts to understand chronic lymphocytic leukemia (CLL) subgroups have focused on multi-omics profiling of patient cohorts (in steady-state) and linking biomarkers to responses. Here, we aim to combine ex-vivo drug screening and gene expression profiling (GEP) in primary CLL samples to dissect disease biology based on the observed phenotype as a qualitative and quantitative function of steady-state features and drug perturbation effects. As most differential gene expression changes in CLL are retrieved from highly expressed genes, we hypothesized that a relevant set of genes could still be reliably captured by reduced sequencing coverage. We profiled five samples with traditional whole transcript TruSeq and 3'-end QuantSeq (Moll et al., Nat Methods, 2014) library preparation with deep and shallow depth sequencing, respectively. We obtained 43.48-72.68 million mappable reads with TruSeq and 2.24-3.78 million with QuantSeq. Although the read coverage was reduced, QuantSeq was able to recover 71.17 ± 0.87% of all genes that were detected by TruSeq with reproducible quantification ( R > 0.85, P < 0.001 for all samples). Shallow sequencing of CLL samples reliably profiled intermediate to highly abundant genes with reduced sensitivity only for lowly expressed genes. We then selected 105 representative CLL patient samples from a well-characterized cohort, treated them ex-vivo with 100 nM ibrutinib for 48h and performed GEP using shallow depth QuantSeq. Sample viability was measured by flow cytometry before and after perturbation. Library preparation and sequencing was successful for 91% of samples. With a median coverage of 2.19 million mappable reads per sample, the principal components (PC) of transcriptional variation in DMSO-treated samples included IGHV (12.0%), methylation subgroup and trisomy 12 status (7.1%). These data show that the screening method reliably captured the transcriptional features of CLL at steady-state and thus support the use of shallow depth RNA sequencing. We were interested in the landscape of transcriptional change after perturbation of the B-cell receptor (BCR) pathway. Samples from the same patients clustered together and spread along a gradient of sample viability in the first PC. After correlation of gene counts from DMSO-treated samples with sample viability and subsequent gene set enrichment analysis, we found activation of the canonical apoptosis pathway via NFκB-mediated TNF-α signaling and cell stress to be associated with this signature of cell death. BTKi-treated samples showed the same signature of spontaneous apoptosis and additionally strong suppression of MYC and OXPHOS activity. To identify the ibrutinib-regulated transcriptional signature, we performed differential gene expression analysis between ibrutinib and DMSO. BTKi treatment induced 904 differentially expressed genes (DEGs) ( FDR < 0.005). These genes were negatively enriched in the hallmarks allograft rejection, MYC targets, TNF-α signaling via NFκB and KEGG pathways associated with BCR signaling, and immune cell response. Top DEGs included e. g. FCRL5, PTPN6, FILIP1L, LRMP and LAPTM5 ( FDR < 0.001) which were also significantly downregulated after in-vivo ibrutinib exposure (Rendeiro et al. Nat Commun, 2020). Inhibition of the BCR induces apoptosis in CLL in an IGHV status-dependent manner. Samples with unmutated IGHV (UM-CLL) were more sensitive to BTKi compared to samples with mutated IGHV (M-CLL) (83.1 vs. 92.3% relative viability, P < 0.001). On gene expression level, we observed more DEGs within UM-CLL (387) compared to M-CLL (182) ( FDR < 0.005). Response to BTKi was largely similar although the effect size in M-CLL tended to be smaller. To identify BCR-dependent genes that were significantly modulated by IGHV, we tested for the interaction between BTKi treatment and IGHV status in the differential expression models. We found the effects on the expression of CA12, FIG RPL5 and P2RY11 to be different upon BTK inhibition between UM- and M-CLL ( FDR < 0.1). In conclusion, we establish shallow depth sequencing in CLL and show it can be used to reliably capture disease biology and drug effects. By treating patient samples with ibrutinib, we show that abrogating BTK strongly suppresses BCR-dependent processes which are largely independent of IGHV status. By adding more pathway perturbations, this approach could be used for further functional dissection of CLL biology.

CDK9 Inhibitor Induces Apoptosis, Autophagy, and Suppression of Tumor Growth in Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a hematopoietic malignancy with a poor prognosis that develops in approximately 5% of human T-cell leukemia virus type 1 (HTLV-1) carriers. Cyclin-dependent kinase 9 (CDK9), together with Cyclin T, forms a transcription elongation factor, positive transcription elongation factor b (P-TEFb). P-TEFb promotes transcriptional elongation by phosphorylating the second serine (Ser2) of the seven amino acid repeat sequence in the C-terminal domain of RNA polymerase II (RNAP II). CDK9 inhibitors suppress cell proliferation by inducing apoptosis in chronic lymphocytic leukemia and breast cancer but there are no reports on autophagy of CDK9 inhibitors. Here, we investigated the effect of LY2857785, a novel CDK9 selective inhibitor, on cell death in ATL-related cell lines in vitro, freshly isolated cells from ATL patients ex vivo, and on ATL tumor xenografts in NOD/SCID mice in vivo. LY2857785 significantly reduced cell viability and induced apoptosis, as shown by annexin V-positive cells, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3, and suppressed the levels of anti-apoptotic protein myeloid cell leukemia-1 (MCL-1). LY2857785 decreased RNAP II Ser2 phosphorylation and downstream c-Myc protein levels. Interestingly, LY2857785 also increased microtubule-associated proteins 1A/1B light chain 3B (LC3)-II binding to autophagosome membranes. Furthermore, LY2857785 decreased the viability of freshly isolated ATL cells and induced apoptosis. Finally, LY2857785 significantly decreased the growth of ATL tumor xenografts. These results suggest that LY2857785 induces cell death of ATL cells by MCL-1-dependent apoptosis and autophagy and has anti-tumor activity.

A Probabilistic Cost-Effectiveness Analysis of Venetoclax and Obinutuzumab as a First-Line Therapy in Chronic Lymphocytic Leukemia in Canada.

Venetoclax is a first-in-class targeted therapy option that is an inducer of apoptosis in chronic lymphocytic leukemia (CLL) cells. The open-label phase III CLL14 clinical trial showed that venetoclax combined with obinutuzumab (VEN+O) is superior to obinutuzumab combined with chlorambucil in newly diagnosed patients with CLL. The aim of this study was to assess the health economic value of VEN+O for the frontline treatment of CLL in Canada from a publicly funded healthcare system perspective. A partitioned survival analyses model was developed including three health states: progression free, progressed, and death. A cycle length of 28 days and a time horizon of 10 years was assumed. VEN+O treatment for a fixed duration of 12 months was compared to obinutuzumab combined with chlorambucil, fludarabine plus cyclophosphamide plus rituximab, bendamustine plus rituximab, chlorambucil plus rituximab, ibrutinib, and acalabrutinib. The population in the model included both unfit and overall frontline CLL patients, two subgroups were also assessed (patients with del17p/TP53 mutations and patients without del17p/TP53 mutations). Survival data extrapolated from the CLL14 trial were used to populate the model. Uncertainty was assessed via one-way sensitivity analyses, probabilistic analyses, and scenario analyses. Based on the probabilistic analyses, unfit frontline CLL patients receiving VEN+O were estimated to incur costs of Canadian dollars ($) 217,727 [confidence interval (CI) $170,725, $300,761] (del17p/TP53: $209,102 [CI $159,698, $386,190], non-del17p/TP53: $217,732 [CI $171,232, $299,063]) and accrue 4.96 [CI 4.04, 5.82] quality-adjusted life-years (del17p/TP53: 3.11 [CI 2.00, 4.20], non-del17p/TP53: 5.04 [CI 4.05, 5.92]). Obinutuzumab combined with chlorambucil, bendamustine plus rituximab, chlorambucil plus rituximab, and ibrutinib accrued lower quality-adjusted life-years and higher costs and as such, VEN+O was the dominant treatment option. The full incremental analysis showed that acalabrutinib was more expensive and more efficacious compared with VEN+O with an incremental-cost-effectiveness-ratio of $2,139,180/quality-adjusted life-year versus VEN+O and not a cost-effective option in Canada. Probabilistic analyses show that at a willingness to pay of $50,000/quality-adjusted life-year gained, VEN+O has the greatest probability of being cost effective. VEN+O is a cost-effective treatment option for unfit frontline CLL patients and provides value for money to healthcare payers.

The pyroptosis-related gene signature predicts prognosis and indicates the immune microenvironment status of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world with great heterogeneity. Pyroptosis has recently been recognized as an inflammatory form of programmed cell death (PCD) and shares a close relationship with apoptosis. Although the role of apoptosis in CLL was comprehensively studied and successfully applied in clinical treatment, the relationship between pyroptosis genes and CLL remained largely unknown. In this study, eight differentially expressed pyroptosis-related genes (PRGs) were identified between CLL and normal B cells. In order to screen out the prognostic value of differentially expressed PRGs, univariate and multivariate Cox regression analyses were conducted and a risk model with three PRG signatures (GSDME, NLRP3, and PLCG1) was constructed. All CLL samples were stratified into high- and low-risk subgroups according to risk scores. The risk model showed high efficacy in predicting both overall survival (OS) and time to first treatment (TTFT). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) showed the dysregulation of immune and inflammatory response in the high-risk group. Single-sample GSEA (ssGSEA) of immune cell infiltration and the activity of immune-related pathways also displayed decreased antitumor immunity in the high-risk group. In conclusion, PRGs are of prognostic value in CLL and may play important roles in tumor immunity, and the underlying relationship between PRGs and CLL needs to be explored further.

PI3K Inhibitors for the Treatment of Chronic Lymphocytic Leukemia: Current Status and Future Perspectives.

Simple SummaryThe development of small agents targeting the B-cell receptor (BCR) pathway revolutionized the treatment of chronic lymphocytic leukemia (CLL). BCR-dependent leukemic cell proliferation is governed by phosphoinositide 3-kinase (PI3K) signaling. The selective PI3Kδ inhibitor idelalisib and dual PI3Kδ/γ inhibitor duvelisib are currently approved by the Food and Drug Administration and European Medicine Agency (only idelalisib) for CLL treatment. Umbralisib, a selective PI3Kδ and casein kinase-1ε (CK1ε) inhibitor, has a different chemical structure and a more favorable safety profile than other PI3K inhibitors (PiK3is); this has enabled its use in combination regimens in clinical trials in first-line and relapsed/refractory CLL. This paper summarizes the development of PI3Kis, their current role and future perspectives in the treatment of patients with CLL.Phosphoinositide 3-kinases (PI3Ks) signaling regulates key cellular processes, such as growth, survival and apoptosis. Among the three classes of PI3K, class I is the most important for the development, differentiation and activation of B and T cells. Four isoforms are distinguished within class I (PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ). PI3Kδ expression is limited mainly to the B cells and their precursors, and blocking PI3K has been found to promote apoptosis of chronic lymphocytic leukemia (CLL) cells. Idelalisib, a selective PI3Kδ inhibitor, was the first-in-class PI3Ki introduced into CLL treatment. It showed efficacy in patients with del(17p)/TP53 mutation, unmutated IGHV status and refractory/relapsed disease. However, its side effects, such as autoimmune-mediated pneumonitis and colitis, infections and skin changes, limited its widespread use. The dual PI3Kδ/γ inhibitor duvelisib is approved for use in CLL patients but with similar toxicities to idelalisib. Umbralisib, a highly selective inhibitor of PI3Kδ and casein kinase-1ε (CK1ε), was found to be efficient and safe in monotherapy and in combination regimens in phase 3 trials in patients with CLL. Novel PI3Kis are under evaluation in early phase clinical trials. In this paper we present the mechanism of action, efficacy and toxicities of PI3Ki approved in the treatment of CLL and developed in clinical trials.

Overexpression of c-Myc-dependent heterogeneous nuclear ribonucleoprotein A1 promotes proliferation and inhibits apoptosis in NOTCH1-mutated chronic lymphocytic leukemia cells.

Background:NOTCH1 mutation is an essential molecular biologic aberration in chronic lymphocytic leukemia (CLL). CLL patients with NOTCH1 mutation have shown an unfavorable survival and a poor response to chemoimmunotherapy. This study aims to present the mechanisms of adverse prognosis caused by NOTCH1 mutation from the perspective of the splicing factor heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).Methods:The microarray data in Gene Expression Omnibus datasets were analyzed by bioinformatics and the function of hnRNPA1 was checked by testing the proliferation and apoptosis of CLL-like cell lines. Afterward, quantitative reverse transcription-polymerase chain reaction and Western blotting were applied to explore the relationship among NOTCH1, c-Myc, and hnRNPA1.Results:RNA splicing was found to play a vital part in NOTCH1-mutated CLL cells; hence, hnRNPA1 was selected as the focus of this study. Higher expression of hnRNPA1 validated in primary NOTCH1-mutated CLL samples could promote proliferation and inhibit apoptosis in CLL. The expression of hnRNPA1 increased when NOTCH1 signaling was activated by transfection with NOTCH1 intracellular domain (NICD)-overexpressed adenovirus vector and declined after NOTCH1 signaling was inhibited by NOTCH1-shRNA. Higher expression of c-Myc was observed in NICD-overexpressed cells and hnRNPA1 expression was downregulated after applying c-Myc inhibitor 10058-F4. Moreover, in NICD-overexpressed cells, hnRNPA1 expression decreased through c-Myc inhibition.Conclusion:Overexpression of c-Myc-dependent hnRNPA1 could promote proliferation and inhibit apoptosis in NOTCH1-mutated CLL cells, which might partly account for the poor prognosis of patients with NOTCH1 mutation.

Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1.

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

Novel Oncogenic Non-Coding RNA：circRIC8B Regulates Lipid Metabolism Via Mir-199b-5p /LPL Axis in Chronic Lymphocytic Leukemia

Objective:During tumor development, energy constraints caused by malnourished microenvironments could exert selective pressure on cancer cells. Tumor cells are driven to metabolic reprogramming to meet the increased demand for energy and metabolites for their rapid proliferation and survival. Chronic lymphocytic leukemia (CLL) is a disease with about 1% of CLL cells proliferating every day which is highly than commonly thought. CLL cells were reported to maintain high levels of proliferation through metabolic changes, but extensive studies did not clearly explain the underlying mechanism of driving genes in CLL metabolism. Circular RNA (circRNA) has recently been shown to play an important role in cell metabolism through lipid accumulation. The purpose of this study is to explore the role of circRNA in lipid metabolism of CLL and provide novel therapeutic targets for CLL.Methods:To analyze circRNAs expression profiles and metabolism map in CLL, peripheral blood mononuclear cells (PBMC) from 53 treatment-naïve CLL patients were collected for transcriptome sequencing. Candidate circRNA circRIC8B in a larger cohort of patients was validated and the clinical characteristics were analyzed. Overexpression and knockdown virus were constructed to infect CLL cells, and untargeted metabolomics was used to find the key lipid metabolic pathway modulating by circRIC8B. The oncogenic functions of circRIC8B were further measured in CLL cell lines (MEC-1 and JVM-3) by performing CCK8 assay, flow cytometry, nile red staining and triglyceride detection. Moreover, we explored the molecular mechanisms of circRIC8B and verified the interactions among circRIC8B, miR-199b-5p and LPL by performing RNA-FISH, RIP, dual-luciferase reporter assay and Western blotting. The killing effects of lipid metabolism inhibitors on CLL cells were detected by CCK8 and flow cytometry.ResultsTranscriptome analysis showed that abnormal lipid metabolism was significantly related to the survival and prognosis of patients with CLL, and circRNAs could be involved in the regulation of lipid metabolism. Kaplan-Meier survival analysis confirmed that patients with higher fatty acid biosynthesis had a significantly lower OS (Figure 1A-B). circRIC8B which is positively correlated with the expression of lipoprotein lipase (LPL) was finally selected for further investigation. qRT-PCR analysis showed that circRIC8B was significantly higher expressed in CLL compared with healthy donors. Moreover, consistent with the sequencing results, circRIC8B was positively correlated with LPL and highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL (Figure 1C). Patients with higher circRIC8B level are associated with worse survival and advanced disease progression (Figure 1D and E). LC-MS/MS results showed that circRIC8B are able to modulated lipid metabolism of CLL cells. Functional analysis demonstrated the promoting role of circRIC8B in cell proliferation. Nile red staining showed lipid accumulation in CLL cells with circRIC8B overexpression increased significantly, while lipid accumulation in circRIC8B knockdown cells decreased significantly, and the quantitative results of triglycerides were similar.Next, we unraveled an original mechanism in CLL that up-regulated circRIC8B was mainly enriched in the cytoplasm, acted as a “sponge” of miR-199b-5p. CCK8 assay, nile red staining showed that the cell viability and lipid accumulating of CLL cell lines were evidently decreased after RNAi of circRIC8B and this result could be reversed by miR-199b-5p inhibitor transfection (Figure 1F-H). In addition, ezetimibe, one of the inhibitors of lipid metabolism was found effectively inhibit the proliferation and promote apoptosis of CLL cells.ConclusionsIn conclusion, as an independent prognostic factor of CLL, circRIC8B was involved in the progress of CLL disease through the miR-199b-5p/LPL axis. In addition, circRIC8B is a key factor in regulating lipid accumulation in CLL, resulting in significant changes in cellular lipid storage, thus supporting the proliferation of CLL cells. Metabolic inhibitor Ezetimibe can effectively block this process and exert anti-tumor functions. This study provides new clues for the role of circRNA in abnormal lipid metabolism of CLL and novel therapeutic strategy for CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunomolecular evaluation of dihydroartemisinin effects on apoptosis in chronic lymphocytic leukemia cell lines

IntroductionDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown to induce apoptosis in many types of cancer cells. In this study, we aimed to determine the effects of DHA on apoptosis in human chronic lymphocytic leukemia (CLL) cell lines. MethodsThe cells were treated separately and combined by DHA and Fludurabine (FLU) during 24, 48 and 72 hours. The cell viabilities determined by XTT method. Following separate and combined treatment of IC50 concentrations of DHA and FLU to the cells during 24 hours, the cells were analyzed by flow cytometry to determine the effects on apopotis staining with AnnexinV FITC and PI. mRNA and protein expression levels of TCTP, Mcl-1, Bcl-2, Bax and Caspase-3 were analyzed to find out the molecular mechanisms of apoptosis by using quantitative real-time PCR and flow cytometric methods. ResultsTreatment with DHA alone or in combination with FLU induced apoptosis in a dose dependent manner in CLL cells. DHA alone was more effective than FLU alone or combined treatment with DHA and FLU. Our results suggest that Bcl-2 protein family member Bax was active in the apoptotic response of CLL cells after DHA treatment. Moreover, the apoptotic response induced by DHA was independent from the p53 mutation status of the CLL cells. ConclusionDHA might be a potential anti-cancer therapeutic for CLL.

PI3Kδ/AKT Inhibition Augments 2-Phenylethynesulfonamide Induced Apoptosis Via the Unfolded Protein Response in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is largely comprised of two major subsets, IGHV unmutated (U-CLL) and mutated (M-CLL) CLL, associated with progressive and indolent disease respectively. Approximately 15% patients harbour a 17p deletion and/or a TP53 mutation which is largely associated with the worst clinical outcome. The development of BCR signalosome inhibitors such as ibrutinib and idelalisib have been instrumental in the treatment of CLL, however these drugs are not curative, only supress the disease and resistance is already developing particularly in patients with a complex karyotype associated with 17p del/ TP53 mutation. Consequently continued development of novel therapies targeting pathways which enhance selective killing of tumor cells independently of p53 are urgently required. We previously demonstrated that 2-phenylacetylenesulfonamide (PAS), induced apoptosis of CLL cells in a p53-independent manner. PAS is suggested to inhibit heat shock protein (HSP) 70, a key protein involved in protein folding. Therefore in CLL cells, PAS would be predicted to cause cellular stress due to the accumulation of misfolded proteins resulting in the activation of the unfolded protein response (UPR). The UPR serves to protect cells from the consequences of protein misfolding and can function in part by activating pro-survival signaling, including the PI3K/AKT pathway. Under normal conditions the ER chaperone BiP inhibits activation of the UPR by binding the three ER transmembrane receptors; inositol-requiring enzyme 1 (IRE-1α), pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Activation of the UPR leads to the upregulation of HSP family members including HSP70 in the cytosol and BiP/GRP78 in the endoplasmic reticulum (ER). These HSPs aid in refolding misfolded proteins or facilitate their degradation via the proteasome. If the stress is resolved, the UPR signalling pathway is switched off; however, if stress persists the UPR remains active and switches from an anti-apoptotic to a pro-apoptotic signal via p38MAPK.Aim: To determine the mechanism of PAS induced apoptosis in CLL cells.Methods: Fifty two CLL samples were treated with PAS (5-20μM) and cellular responses assessed using immunoblotting, flow cytometry and MTT assay.Results: In this study we demonstrated that the UPR is induced in CLL cells following the induction of reactive oxygen species by PAS treatment. This consequently led to the activation of both pro-apoptotic (Noxa, p<0.0001) and anti-apoptotic (AKT, p<0.05) signalling pathways. PAS treatment further increased expression of key stress proteins BiP (p=0.0005) and HSP70 (p=0.001). PAS treatment also resulted in activation of the PERK and IRE-1α arms of the UPR (cleaved XBP-1, phosphorylated eiF2α, phosphorylated p38MAPK) within 2h, which persisted up to 24h. This pathway was pivotal for CLL cell death since small molecule inhibition of either p38MAPK or PERK signalling significantly reduced PAS mediated apoptosis by approximately 50%. However PAS induced apoptosis was simultaneously antagonised by activation of the pro-survival AKT pathway. Consequently we hypothesised that PAS treatment in combination with a PI3Kδ (idelalisib) or AKT (AKTi) inhibitor may promote greater CLL cell apoptosis. Indeed our combination indices (CI) studies demonstrated that simultaneous treatment with an AKTi or the clinically approved PI3Kδ inhibitor idelalisib and PAS synergistically and significantly enhanced apoptosis of CLL cells compared with either agent alone (p<0.05).Conclusion: This data suggests that inhibiting the pro-survival arm of the UPR (PI3K/AKT), whilst simultaneously activating the pro-death arm (HSP70/p38MAPK), may be a useful strategy for the treatment of CLL and particularly samples with a 17p del or TP53 mutation and warrants further clinical investigation particularly in CLL cases that are resistant to BCR kinase or Bcl-2 family inhibitors. DisclosuresStrefford:Roche: Research Funding. Johnson:Novartis: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Carrick: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Karus: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Zenyaku Kogyo: Consultancy, Honoraria; Epizyme: Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele:Gilead: Consultancy, Honoraria; Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding.

First-in-Class Oral Small Molecule Against the Tyrosine Kinase ROR1 (KAN0439834) Induced Significant Apoptosis of Chronic Lymphocytic Leukemia Cells

Background: ROR1 is a transmembrane protein of the receptor tyrosine kinase (RTK) family normally expressed during embryogenesis, but repressed in most normal adult tissues. ROR1 is of importance for cell proliferation, survival, differentiation, metabolism, and polarity. In chronic lymphocytic leukemia (CLL), ROR1 is upregulated and constitutively phosphorylated. Inhibition of ROR1 by siRNA and monoclonal antibodies (mAbs) abrogated downstream kinase activities as well as induced apoptosis of CLL cells. KAN0439834, a small molecule inhibitor (535 Da) was synthesized with the aim to bind to the TK domain of ROR1 and inhibit phosphorylation.Aim: In the present study we investigated the effects of KAN0439834, on survival of CLL cells and signaling, and in vivo effects in NOD-SCID mice xenografted with human CLL cells.Methods: MTT and Annexin V/PI assays were used to analyse cytotoxicity of KAN0439834. Fresh CLL cells were also co-cultured with the human stromal cell line HS-5 (ROR1 negative) and incubated in vitro with KAN0439834 and apoptosis measured (Annexin V/PI). CLL cells were also cultured with KAN0439834 (50-250 nM) for 2 h and recombinant Wnt5a (25-50 µg/mL) was added. The cells were further incubated for 30 min before lysate preparation and cytotoxicity assayed after 24 h. Proximity ligation assay was used to investigate the effects of KAN0439834 on ROR1/LRP6 heterodimerization. Phosphorylated RTKs in CLL cells and the effects of KAN0439834 on phosphorylation of RTKs were analysed by the Human-phospho-RTK array and WB. Finally, in two mice experiments, fresh CLL cells were grafted into NOD-SCID mice and oral KAN0439834 treatment was started 7 days after transplantation and continued for 7 days in the first study and 14 days in the second study.Results: KAN0439834 effectively induced apoptosis of CLL cells (EC50=250 nM) with a 60-fold selectivity for CLL cells compared to normal PBMC. The molecule induced significant apoptosis of CLL cells from all tumor compartments. Importantly, KAN0439834 was significantly more effective than an anti-ROR1 mAb (against the external part of ROR1) in inducing apoptosis of CLL cells. When CLL and HS-5 cells were co-cultured, HS-5 cells could partially prevent induction of apoptosis of CLL cells at low concentrations of KAN0439834, while at higher concentrations of KAN0439834 the presence of stromal cells had no effect. Wnt5a increased phosphorylation of ROR1 in a dose dependent manner. KAN0439834 dephosphorylated both ROR1 and SRC as well as inhibited Wnt5a-induced ROR1 phosphorylation in this system. KAN0439834 dephosphorylated ROR1, LRP6 and associated downstream signaling molecules as GSK3β, PKCδ, MAPK, PI3K, AKT, mTOR, and CREB. KAN0439834 was highly specific for inhibition of ROR1 phosphorylation as compared to other kinase inhibitors in clinical use for cancer treatment. KAN0439834 could be administered in a dose-schedule to mice giving a plasma conc. of > 600 nM for at least 6 h which according to in vitro pharmacokinetic analyses should be sufficient to induce irreversible apoptosis of CLL cells. Oral administration of KAN0439834 to NOD-SCID mice significantly inhibited growth of xenografted human CLL cells. No major side effects were observed in the KAN0439834 treated mice. Immunohistochemistry staining of spleens from the two studies showed a significant decrease of CLL cells in mice treated with KAN0439834 but not in controls.Conclusions: KAN0439834 is the first generation of a novel class of ROR1-inhibiting small molecule drugs developed by phenotypic screening using CLL cells. The small molecule was much more effective in inducing CLL apoptosis than the anti-ROR1 antibody. KAN0439834 inactivated various signaling pathways in the leukemic cells suggested to be associated with ROR1 signaling. The development of new anti-cancer drugs with other mechanisms of action than those clinically available when existing drugs fail is warranted to improve the prognosis of CLL. KAN0439834 may not only be potent in CLL but also in other ROR1 expressing tumors; such studies are in progress. Our results support the further development of ROR1 inhibitors as a new therapeutic principle. DisclosuresHansson:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janssen-Cilag: Honoraria, Research Funding. Vågberg:Kancera AB: Employment. Byström:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Schultz:Kancera AB: Employment. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment. Österborg:Abbvie: Honoraria; Pfizer: Honoraria; Celgene: Research Funding; Gilead: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding. Moshfegh:Kancera AB: Employment.

Modulation of the Tumor Suppressor Protein PP2A Using a Small Molecule Agonist Overcomes Multi-Drug Resistance through Mitochondrial Permeability Transition Pore (MPTP) Dependent Induction of Apoptosis in Chronic Lymphocytic Leukemia

The advent of molecularly targeted therapies has revolutionized the treatment of Non-Hodgkin's Lymphoma (NHL), including Chronic Lymphocytic Leukemia (CLL). Venetoclax (VEN, an inhibitor of Bcl-2) and ibrutinib (IBR, an inhibitor of BTK) generated excellent clinical responses in CLL patients singly and even more effectively in combination (Portell et al. Blood, 2019; Tam et al.NEJM, 2018). Despite its efficacy, the majority of VEN or VEN+IBR responses are partial and resistance usually develops. We previously reported that treatment with microenvironmental agonists ex vivo can generate anti-apoptotic resistance to the VEN+IBR combination in CLL cells via NF-kB-dependent upregulation of multiple anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. Blood Adv, 2017). Here, we report that circulating CLL cells of lymph node origin (CD69positive) exhibit resistance to several BH-domain antagonists (inhibitors of Bcl-2, Mcl-1, and Bcl-xL) when used as single agents. Apoptosis resistance was shown to occur at a pre-mitochondrial level by insufficient activation of Bax/Bak proteins (Fig. 1A-B). Supportive of these findings, ex vivo treatment of primary CLL cells with agonists (sCD40L, TLR9 agonist CpG-ODN, and IL10) known to be expressed in the cancer microenvironment in vivoalso resulted in apoptosis restriction due to defective Bax/Bak activation. Our molecular studies suggest that this resistance is driven by the upregulation of anti-apoptotic proteins, which generates an intracellular environment in which single-drug treatments allow pro-apoptotic proteins (e.g. Bim) to swap between upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2), leading to defective Bax/Bak activation. Hence, therapies aimed at depleting this reservoir must block multiple anti-apoptotic proteins simultaneously or bypass Bax/Bak-dependent apoptosis. Several cancers, including NHL, have evolved mechanisms to suppress the activity of Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase known to regulate cell survival/proliferation. In our current study involving various cancer cell lines (~250), a small molecule agonist of PP2A (SMAP, TRC-382) showed broad activity across blood cancer cell lines. A further pharmacologically optimized SMAP compound (DT061), also known to be safe in animal models (Tohmé et al. JCI Insight, 2019), was effective even in blood cancer cell lines and agonist treated CLL samples resistant to several BH-domain antagonists (inhibitors on Mcl-1, Bcl-xL, and Bcl-2), suggesting that PP2A activation could overcome pre-mitochondrial apoptosis restriction. DT061 was able to overcome drug resistance in patient-derived CD69positive CLL cells through the induction of Bax/Bak-independent apoptosis (Fig. 1C). Supportive of this finding, DT061 was also able to induce apoptosis in Bax/Bak double knockout CLL cell line (MEC1) (Fig. 1D). To determine the mechanisms underlying SMAP-induced apoptosis, we examined additional pathways capable of triggering apoptosis such as mitochondrial permeability transition pore (MPTP), calcium channels, and VDAC1 using selected small molecule inhibitors. Only inhibitors blocking MPTP (NIM811 or cyclopsorin-A/CspA) significantly impaired the DT061- but not VEN-induced apoptosis in primary CLL cells (Fig. 2A-B). Additionally, our analysis using the CalceinAM/CoCl2 method revealed that DT061 was able to induce MPTP opening in primary CLL cells, which was inhibited in the presence of NIM811 or CspA, suggesting that DT061 induces apoptosis through MPTP activation (Fig. 2C). In summary, we identify, in the circulation of treatment-naïve and treated CLL patients, microenvironmentally-activated leukemic B cells that exhibit apoptosis resistance at the level of Bax/Bak activation. Therapies aimed at depleting this reservoir of drug-resistant leukemic B cells must trigger a process of Bax/Bak independent apoptosis. We demonstrate that PP2A activation using the SMAP (DT061) induces apoptosis in drug resistant CLL cells bypassing the Bax/Bak pathway. The apoptosis induction was dependent on MPTP activation. Collectively, this work highlights the existence of an anti-apoptotic multi-drug resistant pool of CLL cells in patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Disclosures Williams: Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapuetics: Research Funding; Celgene: Honoraria; Gilead: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Xian Janssen: Honoraria. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Narla:The Icahn School of Medicine at Mount Sinai: Patents &amp; Royalties: nternational Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2; Mount Sinai: Other: Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology.; RAPPTA Therapeutics: Consultancy, Current equity holder in private company.

DAP Kinase-Related Apoptosis-Inducing Protein Kinase 2 (DRAK2) Is a Key Regulator and Molecular Marker in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-κB, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis.

Bifunctional Therapeutic Peptides for Targeting Malignant B Cells and Hepatocytes: Proof of Concept in Chronic Lymphocytic Leukemia

AbstractProtein–protein interactions are well recognized as therapeutic targets and therefore interfering peptides (IP) that block these interactions are receiving increasing attention. Four different tumor‐penetrating peptides (TPPs) (iRGD, RPARPAR, Linear TT1 (LinTT1), and cyclic TT1 (TT1)) are associated to an IP that blocks the interaction between the protein phosphatase PP2A and its binding protein SET, generating new bifunctional peptides able to intracellularly target the PP2A/SET interaction in malignant B cells and tumoral hepatocytes. The TPPs are able to penetrate into B cells of patients suffering chronic lymphocytic leukemia (CLL) and into tumoral hepatocytes but not into B cells from healthy donors and healthy hepatocytes. The association of cargo does not affect the penetration of the TPPs in CLL B cells. All the bifunctional peptides induce apoptosis in human CLL B cells and tumoral hepatocytes, and stability tests reveal that iRGD‐IP, RPARPAR‐IP, and TT1‐IP are stable after 24 h incubation in human serum. The iRGD associated with the IP significantly increases the survival of mice bearing xenograft models of CLL without any symptom of toxicity, suggesting that the bifunctional peptides may have a therapeutic application for selective tumoral targeting of PP2A/SET interaction, which is deregulated in several cancers, including CLL.