Apoptosis In Chronic Lymphocytic Leukemia Cells Research Articles

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

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

Disrupting pro-survival and inflammatory pathways with dimethyl fumarate sensitizes chronic lymphocytic leukemia to cell death

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells.

Curcumin Enhances Ibrutinib Induced Killing Effect Against TP53-Mutated Chronic Lymphocytic Leukemia Cells in Vitro

Introduction TP53 mutation and del (17p) are the most important poor prognostic factors in patients with Chronic Lymphocytic Leukemia (CLL).Patients with deletion 17p or TP53 mutations tend to have lower response rates to standard first-line chemoimmunotherapy and a more aggressive clinical course(Stilgenbauer et. al. Blood 2014). RESONATE-2 study long-term follow-up showing that ibrutinib continues to demonstrate significant and durable clinical benefit in older patients, including those with TP53 mutation(Barr et. al. Blood Adv 2022).However, CLL bearing del(17p) showed inferior OS and time to next treatment compared to non-del(17p)(Mato et. al. Haematologica 2022). A significant proportion of patients treated with single-agent ibrutinib experienced CLL progression.BTK and/or PLCG2 mutations were present in 80.0% of patients progressing with CLL, confirming prior reports on the high prevalence of these mutations in patients with ibrutinib-resistant CLL(Ahn et. al. Blood 2017). Curcumin is a bioactive natural polyphenol compound extracted from the Curcuma longa(Zingiberaceae plant). Curcumin has anti-tumor effect and can induce apoptosis and inhibit the proliferation of a series of tumor cells(Willenbacher et. al. Int J Mol Sci 2019). Curcumin may limit the development of therapy-related toxicity and reduce the financial burden of treatment.In this study, we found that Curcumin inhibits TP53-mutated CLL cells, and eliciting a strong synergistic cytotoxic effect in combination with ibrutinib. Methods The inhibitory effect of ibrutinib combined with curcumin on the proliferation of TP53-mutated Chronic Lymphocytic Leukemia cells was detected by CCK8, and the apoptosis of TP53-mutated CLL cells induced by ibrutinib combined with curcumin was detected by flow cytometry;The differences of gene and signal pathway expression among different treatment groups were analyzed by RNA-seq ;Using Western blot technology to verify the expression of related pathway proteins and explore the mechanism of curcumin increasing the killing effect of ibrutinib against TP53-mutated Chronic Lymphocytic Leukemia cells. Results The CCK8 results showed that the IC50 of ibrutinib for 24 hours was 40.61±2.35μM, while the IC50 of curcumin for 24 hours was 26.6±0.94μM. The IC50 of ibrutinib for 48 h was 14.6 ±0.8307μM, and the IC50 of curcumin for 48 h was 15.7±2.53μM.Cell inhibition of various group for 48h is shown in Figure 1.The CI of ibrutinib combined with curcumin at 24 hours and 48 hours was calculated by CompuSyn software. The results showed that the CI values of ibrutinib combined with curcumin were less than 1 at 24 h and 48 h, suggesting that ibrutinib combined with curcumin has a synergistic killing effect on CLL cells with TP53 mutation;Flow cytometry showed that the IC50 of ibrutinib for 24 hours was 67.20±2.86μM, while the IC50 of curcumin for 24 hours was 144±6.6 μM. The CI value of ibrutinib combined with curcumin was less than 1 at 24 h, suggesting that ibrutinib combined with curcumin has a synergistic promoting apoptosis on CLL cells with TP53 mutation;RNA-seq analysis and KEGG analysis of down-regulated genes of combination group compared with the control group were mainly enrich in PI3K/AKT, Cytokine-Cytokine Recptor signal pathway and so on;4.The results of Western blot showed that the protein expression levels of P65PI3K and p53 in the single drug group and the combination group were down-regulated in varying degrees, especially in the combination group（Figure 2). Conclusion Ibrutinib combined with curcumin has synergistic killing effect on TP53- mutated CLL cells;Curcumin increases the killing effect of ibrutinib on TP53-mutated CLL cells by inhibiting PI3K and NFκB pathway, and degrading p53mt protein.

The Unique BCR Inhibiting Properties of BMS-986205 in Chronic Lymphocytic Cells

B cell receptor (BCR) signaling is recognised as a central pathway in the pathogenesis of chronic lymphocytic leukaemia (CLL), the most common leukemia in the western world. In CLL, the BCR pathway is activated by antigens in the tissue microenvironment to promote the maintenance and expansion of leukemic cells. The impressive inhibition of clonal expansion of CLL cells and the disease control achieved with tyrosine kinase inhibitors that block BCR signaling provided further evidence of the role of BCR in CLL. Indoleamine 2,3-dioxygenase 1 (IDO1) is the inducible and rate-limiting enzyme involved in the catabolism of tryptophan to kynurenine. It was discovered as a modulator of the innate immune response during infection. Subsequent discoveries have implicated IDO1 as a player in acquired immune tolerance. IDO1 activity has been identified in several types of cancer cells as well as in their tumor-surrounding microenvironment. We have previously investigated and characterized the impact of IDO1 expression in CLL. Our results show that CLL microenvironmental stimuli positively modulate IDO1 expression in leukemic cells and that increased IDO1 activity leads to sustained survival of malignant lymphocytes and impaired drug sensitivity through the autocrine/paracrine activation of the aryl hydrocarbon receptor by kynurenine (Atene CG et al. 2022). To assess the functional and clinical significance of IDO1 enzymatic activity in CLL, we used several inhibitors that are currently in late-stage clinical trials. Using BMS-986205 (BMS), an irreversible inhibitor of IDO1 with the best cell-based potency, in primary patient cells, we observed not only a reduction in kynurenine production due to enzymatic inhibition of IDO1, but also lower levels of IDO1 RNA and protein following BCR stimulation with α-IgM. As we have previously shown that BCR activation can induce IDO1 expression, we treated CLL cells with increasing doses of BMS for 3 hours and then added α-IgM to the culture. BMS is able to counteract the pro-survival effect of BCR triggering, leading to significant apoptosis of CLL cells, while the viability of CLL cells in the absence of external stimuli was not significantly modulated. Focusing on the complex signaling cascade activated by BCR engagement, we observed that BMS impaired the phosphorylation of upstream kinases such as SYK and BTK, but also CD79a, CD19 and BLNK, in addition to downstream effectors such as AKT. We therefore investigated which factors that negatively regulate the BCR pathway are modulated by BMS treatment. By analyzing both mRNA and protein levels, we measured a significant induction of the phosphatases PTPN6, PTPN22 and SHIP1. Since BMS also showed a downregulation of total SYK expression, we are analyzing the expression of the proto-oncogene CBL, an E3 ubiquitin ligase known to be involved in the negative regulation of SYK. Further studies should be undertaken to determine whether, in addition to its role as an IDO1 inhibitor, BMS may also have the ability to indirectly modulate and disrupt BCR signaling. Efforts are underway to identify the proteome-wide direct target of BMS in CLL cells. In conclusion, our limited and preliminary data suggest that BMS could be considered as a potential therapeutic strategy to target CLL cells with a double hit: by blocking the pro-survival effect of BCR signaling, one of the driving forces for CLL cell survival and proliferation, and by inhibiting IDO1/kynurenine action to restore sensitivity to key leukemia treatments.

A CX3CR1 (fractalkine receptor) Small Molecular Antagonist (KAND567) Suppressed the Growth Promoting Effect of Monocytes on Chronic Lymphocytic Leukemia Cells

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia worldwide. Novel treatment strategies are needed to improve the prognosis. Non-malignant cells of the tumor microenvironment (TME) are of importance for the growth of tumor cells. Monocytes expressing various chemokine receptors as CX3CR1 (the fractalkine receptor (FKNR)) have been shown to promote the growth of tumor cells in vitro and in vivo. In CLL, monocyte derived nurse-like cells (NLC) support the growth of CLL cells, promoting leukemic cell proliferation and survival by secretion of chemokines/cytokines. In this study, we studied the role of autologous monocytes on the survival of CLL cells and the effects of a small molecule antagonist of CX3CR1 (KAND567/FKNRa). KAND567 is in clinical trials for diseases with an activated fractalkine system as part of the pathobiology including covid-19 infected patients as well as patients with acute myocardial infarction and ovarian carcinoma. Methods: Primary CLL cells and B cells (CD19 +) of healthy donors as well as autologous monocytes (CD14 +) from CLL patients (n=22) and healthy control donors (n=11) were cultured alone or in co-cultures (isolated B cells together with autologous monocytes) with the FKNRa (250 and 1000 nM) for up to 120 h. Apoptosis of CD19 + cells and monocytes was analysed by Annexin V/PI staining, Western blotting and the IncuCyte® Live Cell Imaging System. The NBT assay was also applied to check morphological changes of monocytes. Plasma concentration of fractalkine (CX3CL1) was determined by ELISA. Results and discussion: CLL patients had a significant (p=0.0001) increase in the plasma concentration of CX3CL1 compared to healthy controls. CLL cells as well as normal B cells did not express surface CX3CR1. However, monocytes from both CLL patients and healthy donors expressed high levels of surface CX3CR1. A significantly higher proportion of pro-inflammatory intermediate (CD14 +/CD16 +) and non-classical (CD14 -/CD16 +) monocytes (p<0.0001 and <0.01 resp.) as well as intermediate and non-classical CX3CR1 + monocytes were observed in patients with active CLL compared to healthy donors (p<0.0001 and <0.01 resp.). Moreover, CX3CR1 + intermediate and non-classical monocytes were significantly higher in CLL patients with active disease compared to those with early-stage disease (p<0.0001 and <0.001 resp.). A growth supportive effect of autologous monocytes was observed on survival of both CLL cells and healthy donor B cells in 120 h cell co-culture experiments (p<0.001 and <0.01 resp.). FKNRa reduced the number of alive CLL cells in a dose-dependent manner in co-cultures with autologous monocytes (p<0.05). No effect was seen on CD19 + B cells of healthy donors co-cultured with autologous monocytes. FKNRa did not induce apoptosis of CLL cells alone. Furthermore, FKNRa also inhibited transformation of CLL monocytes to NLC (p<0.05) which was not the case for normal monocytes. Conclusion: The CX3CR1/CX3CL1 (fractalkine receptor/ligand)) axis appears to be activated in CLLand might be of importance for the NLC-driven growth of CLL cells. This is the first report of a small molecule antagonist of CX3CR1 reducing the growth supportive effect of autologous CLL monocytes on leukemic CLL cells. Disruption of growth promotion induced by monocytes may prevent leukemic cell proliferation and survival. Inhibition of autologous monocytes in the TME might represent a new therapeutic strategy in CLL, complementary to treatment with drugs that directly target the tumor cells.

NOD2 silencing promotes cell apoptosis and inhibits drug resistance in chronic lymphocytic leukemia by inhibiting the NF-κB signaling pathway.

Recent years have witnessed increasing studies on the effect of epigenetic silencing of genes in the progression of chronic lymphocytic leukemia (CLL). This study investigates whether the nucleotide binding oligomerization domain containing 2 (NOD2) participates in the cell apoptosis and drug resistance of CLL cells. Cells were treated with adriamycin (ADR), etoposide, aclacinomycin and daunorubicin. After treatment, drug resistance and cell proliferation were examined to detect the inhibitory effect of ADR on cell proliferation; flow cytometry to identify ADR accumulation, the cell cycle distribution and apoptosis after transfection, and rhodamine 123 accumulation and efflux tests to assess P-glycoprotein (P-gp) function. NOD2 silencing or inhibition of the nuclear factor kappa-B (NF-κB) signaling pathway suppressed the multidrug resistance level in CLL, the inhibition rate, and cell proliferation caused by ADR at concentrations of approximately 0.25-1.5 μmol/L. Greater accumulation of ADR was observed in the CLL-AAT cell line than in the CLL-AAT/A02 cell line, but NOD2 silencing or inhibition of the NF-κB signaling pathway further increased the accumulation of ADR drugs in the CLL-AAT cell line and inhibited the drug efflux pump function of P-gp. Additionally, NOD2 silencing or NF-κB signaling pathway inhibition increased the apoptotic rate. The results of this study indicate that NOD2 promotes cell apoptosis and reduces the drug resistance of CLL by inhibiting the NF-κB signaling pathway.

Focal adhesion kinase activation by calcium-dependent calpain is involved in chronic lymphocytic leukaemia cell aggressiveness.

Signalling events downstream the B-cell receptor (BCR) are central for the survival and progression of chronic lymphocytic leukaemia (CLL) cells. Focal adhesion kinase (FAK), regulated through calpain, interacts with molecules of BCR signalling, cytoskeletal modelling and disease progression, such as Src/Lyn, cortactin and HS1. Hypothesizing that FAK might play a key role in CLL pathogenesis, we observed a down-modulation of FAK whole form, associated with FAK cleavage due to calpain activity upon BCR stimulation. Patients, whose cells were able to release Ca++ after BCR stimulation, had less amount of full-length FAK, which translated into a higher presence of cleaved/activated form of the protein phosphorylated at Y397, these features being mostly shown by immunoglobulin heavy chain (IGHV)-unmutated poor-prognosis patients. Moreover, we found that cortactin and HS1 proteins were overexpressed in those cells, suggesting a possible interplay with FAK. Treatment with the FAK inhibitor Defactinib was able to induce apoptosis in CLL cells. In conclusion, the malignant phenotype in unfavourable-prognosis patients seems to be encouraged by the overexpression of cortactin and HS1, that, together with FAK, may be involved in a druggable pathogenetic pathway in CLL.

PP2A modulation overcomes multidrug resistance in chronic lymphocytic leukemia via mPTP-dependent apoptosis.

Targeted therapies such as venetoclax (VEN) (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple antiapoptotic proteins and display resistance to proapoptotic agents. Here, we demonstrated that multidrug-resistant CLL cells in vivo exhibited apoptosis restriction at a pre-mitochondrial level due to insufficient activation of the Bax and Bak (Bax/Bak) proteins. Co-immunoprecipitation analyses with selective BH domain antagonists revealed that the pleiotropic proapoptotic protein (Bim) was prevented from activating Bax/Bak by "switching" interactions to other upregulated antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that small-molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug-resistant leukemia and lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug-resistant CLL cells through induction of mitochondrial permeability transition pores, independent of Bax/Bak. DT-061 inhibited the growth of wild-type and Bax/Bak double-knockout, multidrug-resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug-resistant CLL cells in patients and validated a pharmacologically tractable pathway to deplete this reservoir.

Prognostic value and therapeutic targeting of XPO1 in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a subtype of B-cell malignancy with high heterogeneity. XPO1 is highly expressed in many hematological malignancies, which predicts poor prognosis. In the study, we aimed to explore the prognostic role of XPO1 and the therapeutic effect of Selinexor, a selective inhibitor of nuclear export, which targets XPO1. We collected 200 CLL samples in our center to confirm XPO1 mRNA expression and analyzed the correlation between XPO1 expression and prognosis. Then, we decreased XPO1 expression with Selinexor to explore the effect of proliferation inhibition, cell cycle arrest, and apoptosis in CLL cell lines. RNA-Seq was performed to explore potential mechanisms. We analyzed XPO1 expression in a cohort of 150 treatment naive patients and another cohort of 50 relapsed and refractory (R/R) patients and found that XPO1 expression was upregulated in 76% of CLL patients compared with healthy donors. Survival analysis suggested that patients with increased XPO1 expression had inferior treatment-free survival (P = 0.022) and overall survival (P = 0.032). The inhibitor of XPO1, Selinexor, induced apoptosis in primary CLL cells. We showed the effects of Selinexor on proliferation inhibition, cell cycle arrest, and apoptosis in CLL cell lines with JVM3, MEC1, and ibrutinib-resistant (MR) cells via nuclear retention of cargo proteins of IκBα, p65, p50, and FOXO3a. Moreover, downregulation of the NF-κB and FOXO pathways was a common feature of the three CLL cell lines responding to Selinexor, indicating the potential application of XPO1 inhibitor even in the high-risk CLL cells. We identified XPO1 as an unfavorable prognostic factor for CLL patients and provided a rationale for further investigation of the clinically XPO1 targeted therapeutic strategy against CLL.

PLX51107 Enhances the Anti-Tumor Activity of Targeted Therapy in Chronic Lymphocytic Leukemia When Combined with Venetoclax

Background: The development of drugs targeting BTK and BCL2 have dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents have been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulate gene transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of BRD4i PLX51107, and their cooperative role with BCL2 inhibitor Venetoclax in CLL. Methods: 56 primary CLL cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults using Roswell Park Comprehensive Cancer Center protocol CIC-01-16. Peripheral blood mononuclear cells (PBMC) were seeded at 3 x106/mL in a 384 or 6 well tissue culture plates and exposed to different doses of PLX51107 (1-20uM) as a single agent or in combination with Venetoclax (1-100nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. Anti-tumor effect was also analyzed by induction of apoptosis using Annexin-V/PI staining by flow cytometry at 72hrs. Cells pellets were saved for western blotting to determine expression of BRD4 members and different members of the B-cell receptor, NF-kB pathway as also the BCL-2 family of proteins. Bcl-2 family profiling was performed via Milliplex Bcl-2 family protein panels 1 and 2 to look at overall changes in Bcl-2 family expression. Results: Single agent exposure to PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells (Figure 1a). Next, we examined the interaction between the PLX51107 and Venetoclax. In 72hr exposure the combination of PLX51107 with Venetoclax with an ATM deletion was synergistic in 4 of 8 samples and 4 of 7 samples with p53 deletion. Annexin/PI staining yielded apoptotic cell death in the single agent and combination exposure with Venetoclax. Western blotting showed a modulation of both BRD4 proteins as well as Bcl-2 family member proteins (Figure 1b). Bcl-2 family profiling via Milliplex panels demonstrated the ratio of Bcl-2/Bcl x/L was much higher in samples with a high Venetoclax IC50 (136/1) compared to Venetoclax low IC50 (54/1). (Figure 1) -A) Samples with p53 deletion and corresponding synergy. (B) BCL2 family protein expression of CLL sample 214c. Conclusions: Our data indicates that a) PLX51107 has an antitumor effect in CLL cells; b) the combinational treatment of Venetoclax with PLX51107 highly improved the apoptotic effect of single agent treatments. A better understanding of the key signaling pathways for CLL cells' proliferation and survival impacted by BRD4i in combination with Venetoclax would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ezrin is highly expressed and a druggable target in chronic lymphocytic leukemia

AimsDespite the development of therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors and plays a key role in cell survival and BCR-mediated signaling activation in B-cell lymphomas. However, its role in hematological neoplasms remains poorly explored. Main methodsThe present study assessed EZR expression in samples from CLL patients and healthy donors and evaluated the cellular and molecular effects of a pharmacological EZR inhibitor, NSC305787, in CLL cellular models. Key findingsEZR was highly expressed and positively associated with relevant signaling pathways related to CLL development and progression, including TP53, PI3K/AKT/mTOR, NF-κB, and MAPK. NSC305787 reduced viability, clonogenicity, and cell cycle progression and induced apoptosis in CLL cells. Pharmacological EZR inhibition also attenuated ERK, S6RP, and NF-κB activation, indicating that EZR not only associates with but also activates these signaling pathways in CLL. Ex vivo assays revealed that the EZR inhibition-induced cell viability reduction was independent of molecular risk and the Binet stage. SignificanceOur study provides insights into EZR as a pharmacological target in CLL, shedding light on a novel strategy for treating this disease.

Inhibition of p38 MAPK or immunoproteasome overcomes resistance of chronic lymphocytic leukemia cells to Bcl-2 antagonist venetoclax

Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in CLL, the development of resistance diminishes their therapeutic activity. This is also true for the Bcl-2 antagonist venetoclax. We investigated the molecular mechanisms that drive venetoclax resistance in CLL, with a clear focus to provide new strategies to successfully combat it. Activation of CLL cells with IFNγ, PMA/ionomycin, and sCD40L diminished the cytotoxicity of venetoclax. We demonstrated that the metabolic activity of cells treated with 1 nM venetoclax alone was 48% of untreated cells, and was higher for cells co-treated with IFNγ (110%), PMA/ionomycin (78%), and sCD40L (62%). As of molecular mechanism, we showed that PMA/ionomycin and sCD40L triggered translocation of NFκB in primary CLL cells, while IFNγ activated p38 MAPK, suppressed spontaneous and venetoclax-induced apoptosis and induced formation of the immunoproteasome. Inhibition of immunoproteasome with ONX-0914 suppressed activity of immunoproteasome and synergized with venetoclax against primary CLL cells. On the other hand, inhibition of p38 MAPK abolished cytoprotective effects of IFNγ. We demonstrated that venetoclax-resistant (MEC-1 VER) cells overexpressed p38 MAPK and p-Bcl-2 (Ser70), and underexpressed Mcl-1, Bax, and Bak. Inhibition of p38 MAPK or immunoproteasome triggered apoptosis in CLL cells and overcame the resistance to venetoclax of MEC-1 VER cells and venetoclax-insensitive primary CLL cells. In conclusion, the p38 MAPK pathway and immunoproteasome represent novel targets to combat venetoclax resistance in CLL.

Hes1 Controls Proliferation and Apoptosis in Chronic Lymphoblastic Leukemia Cells by Modulating PTEN Expression.

Hairy and enhancer of split homolog-1 (HES1), regulated by the Notch, has been reported to play important roles in the immune response and cancers, such as leukemia. In this study, we aim to explore the effect of HES1-mediated Notch1 signaling pathway in chronic lymphocytic leukemia (CLL). Reverse transcription quantitative polymerase chain reaction and Western blot assay were conducted to determine the expression of HES1, Notch1, and PTEN in B lymphocytes of peripheral blood samples of 60 CLL patients. We used lentivirus-mediated overexpression or silencing of HES1 and the Notch1 signaling pathway inhibitor, MW167, to detect the interaction among HES1, Notch1, and PTEN in CLL MEC1 and HG3 cells. MTT assay and flow cytometry were employed for detection of biological behaviors of CLL cells. HES1 and Notch1 showed high expression, but PTEN displayed low expression in B lymphocytes of peripheral blood samples of patients with CLL in association with poor prognosis. HES1 bound to the promoter region of PTEN and reduced PTEN expression. Overexpression of HES1 activated the Notch1 signaling pathway, thus promoting the proliferation of CLL cells, increasing the proportion of cells arrested at the S phase and limiting the apoptosis of CLL cells. Collectively, HES1 can promote activation of the Notch1 signaling pathway to cause PTEN transcription inhibition and the subsequent expression reduction, thereby promoting the proliferation and inhibiting the apoptosis of CLL cells.

STAT3 Activates the Pentraxin 3 Gene in Chronic Lymphocytic Leukemia Cells.

Pentraxin-related protein 3 (PTX3), commonly produced by myeloid and endothelial cells, is a humoral pattern recognition protein of the innate immune system. Because PTX3 plasma levels of patients with chronic lymphocytic leukemia (CLL) are high and most circulating cells in patients with CLL are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from seven of seven patients with CLL produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes coexpressing CD19 and CD5, and confocal microscopy showed that PTX3 is present in the cytoplasm of CLL cells. Because STAT3 is constitutively activated in CLL cells, and because we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 Abs precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an EMSA, we established and used a dual-reporter luciferase assay to confirm that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 enhanced apoptosis of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

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.

Perspectives on Precision Medicine in Chronic Lymphocytic Leukemia: Targeting Recurrent Mutations-NOTCH1, SF3B1, MYD88, BIRC3.

Chronic lymphocytic leukemia (CLL) is highly heterogeneous, with extremely variable clinical course. The clinical heterogeneity of CLL reflects differences in the biology of the disease, including chromosomal alterations, specific immunophenotypic patterns and serum markers. The application of next-generation sequencing techniques has demonstrated the high genetic and epigenetic heterogeneity in CLL. The novel mutations could be pharmacologically targeted for individualized approach in some of the CLL patients. Potential neurogenic locus notch homolog protein 1 (NOTCH1) signalling targeting mechanisms in CLL include secretase inhibitors and specific antibodies to block NOTCH ligand/receptor interactions. In vitro studies characterizing the effect of the splicing inhibitors resulted in increased apoptosis of CLL cells regardless of splicing factor 3B subunit 1 (SF3B1) status. Several therapeutic strategies have been also proposed to directly or indirectly inhibit the toll-like receptor/myeloid differentiation primary response gene 88 (TLR/MyD88) pathway. Another potential approach is targeting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inhibition of this prosurvival pathway. Newly discovered mutations and their signalling pathways play key roles in the course of the disease. This opens new opportunities in the management and treatment of CLL.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Abstract 937: The PP2A activation using a small molecule agonist triggers apoptosis by releasing mitochondrial permeability transition pores in multi-drug resistant leukemic B cells

Abstract The Bcl-2 inhibitor venetoclax (VEN) shows clinical activity in Chronic Lymphocytic Leukemia (CLL), and the response is potentiated when combined with the BTK inhibitor ibrutinib (IBR) (Portell et al. 2019; Tam et al. 2018). However, many VEN or VEN+IBR responses are incomplete and drug resistance develops in most cases. We previously noted that CLL cells exposed to microenvironmental agonists ex vivo exhibit resistance to VEN+IBR due to upregulation of multiple anti-apoptotic proteins (Jayappa et al. 2017). Here, we find in the circulation of CLL patients that activated (CD69Pos) leukemic B cells recently emigrated from the lymph node exhibit resistance to inhibitors of Bcl-2, Mcl-1, or Bcl-xL due to impaired activation of Bax/Bak proteins. Our molecular analysis suggests that multiple upregulated anti-apoptotic proteins (Mcl-1, Bcl-xL, and Bcl-2) swap for pro-apoptotic proteins (e.g. Bim) in these cells when subjected to single-drug treatments, leading to defective Bax/Bak activation resulting in resistance to drug induced apoptosis. A large-scale cell-based screen using small molecule agonist of Protein Phosphatase 2A (PP2A) (SMAP, TRC-382) revealed that blood cancer cell lines are highly sensitive to SMAP mediated activation of PP2A, a serine/threonine phosphatase known to regulate cell survival. A further pharmacologically optimized SMAP compound (DT061) showed activity even in leukemia cell lines or patient-derived CD69Pos CLL cells resistant to inhibitors of Bcl-2, Mcl-1, or Bcl-xL, suggesting PP2A activation can overcome apoptosis resistance. The PP2A activator DT061 induced apoptosis in these cells in the absence of Bax/Bak activation and was equally effective in an isogenic Bax/Bak double knockout cells, suggesting PP2A activation overcomes multi-drug resistance via induction of Bax/Bak-independent apoptosis. We next examined various Bax/Bak-independent apoptosis mechanisms using inhibitors, and found that inhibitors of mitochondrial permeability transition pores (mPTP) (NIM811 and cyclopsorin-A) blocked the DT061-induced apoptosis in CLL cells. Using the CalceinAM/CoCl2 assay, we noted that DT061 triggers mPTP opening in primary CLL cells, which was blocked by mPTP inhibitors, suggesting DT061 induces apoptosis via mPTP activation. In summary, microenvironmentally activated cancer cells displaying resistance to apoptosis due to defective Bax/Bak activation are found de novo in CLL patients. Reactivation of the tumor suppressive serine/threonine phosphatase by a series of small molecules (SMAPs) overcomes this resistance by inducing mPTP dependent apoptosis. Collectively, these findings demonstrate the existence of an anti-apoptotic multi-drug resistant pool of cancer cells in CLL patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Citation Format: Kallesh D. Jayappa, Caroline Farrington, Vicki L. Gordon, Shekhar Saha, Christopher Morris, Krista M. Isaac, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Goutham Narla, Michael J. Weber. The PP2A activation using a small molecule agonist triggers apoptosis by releasing mitochondrial permeability transition pores in multi-drug resistant leukemic B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 937.

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.