Apoptosis In Chronic Lymphocytic Leukemia Research Articles

A feedback circuit of miR-34a/MDM4/p53 regulates apoptosis in chronic lymphocytic leukemia cells.

BackgroundDysfunction of apoptosis is a significant characteristic in chronic lymphocytic leukemia (CLL). Murine double minute 4 (MDM4), miR-34a and TP53 are found to participate in modulating cellular apoptosis while the specific mechanism keeps unclear. This study was designed to investigate the potential feedback circuit among MDM4, miR-34a and TP53.MethodsAccording to the bioinformatic approaches, there are 4 miR-34a candidate binding domains in MDM4. Use dual luciferase reporter gene to verify the regulation between miR-34a and MDM4. Flow cytometry was used to detect the change of apoptosis level in CLL cells before and after miR-34a mimics and shMDM4 were respectively transfected into primary CLL cells in vitro. Meanwhile, Real-time PCR was used to detect the change of RNA expression of MDM4, miR-34a and TP53.ResultsUp-regulated expression of miR-34a or down-regulated expression of MDM4 could increase apoptosis of CLL cells, inhibit expression of MDM4 and decrease expression of p53 in mRNA level compared to negative control (NC) or shNC (P<0.05). The luminescence of psiCHECK-2-MDM4 EXON 11 can be effectively inhibited by miR-34a (P<0.05).ConclusionsMiR-34a could modulate MDM4 by binding to MDM4 exon 11 instead of 3’UTR. This research thus highlights a forceful evidence for miR-34a/MDM4/p53 feedback circuit in CLL apoptosis.

Relation of Neutrophil Gelatinase-Associated Lipocalin Overexpression to the Resistance to Apoptosis of Tumor B Cells in Chronic Lymphocytic Leukemia.

The resistance to apoptosis of chronic lymphocytic leukemia (CLL) cells partly results from the deregulated production of survival signals from leukemic cells. Despite the development of new therapies in CLL, drug resistance and disease relapse still occur. Recently, neutrophil gelatinase-associated lipocalin (NGAL), a secreted glycoprotein, has been suggested to have a critical role in the biology of tumors. Thus, we investigated the relevance of NGAL in CLL pathogenesis, analyzed the expression of its cellular receptor (NGAL-R) on malignant B cells and tested whether CLL cells are resistant to apoptosis through an autocrine process involving NGAL and NGAL-R. We observed that NGAL concentrations were elevated in the serum of CLL patients at diagnosis. After treatment (and regardless of the therapeutic regimen), serum NGAL levels normalized in CLL patients in remission but not in relapsed patients. In parallel, NGAL and NGAL-R were upregulated in leukemic cells from untreated CLL patients when compared to normal peripheral blood mononuclear cells (PBMCs), and returned to basal levels in PBMCs from patients in remission. Cultured CLL cells released endogenous NGAL. Anti-NGAL-R antibodies enhanced NGAL-R+ leukemia cell death. Conversely, recombinant NGAL protected NGAL-R+ CLL cells against apoptosis by activating a STAT3/Mcl-1 signaling pathway. Our results suggest that NGAL and NGAL-R, overexpressed in untreated CLL, participate in the deregulation of the apoptotic machinery in CLL cells, and may be potential therapeutic clues for CLL treatment.

In Chronic Lymphocytic Leukemia the JAK2/STAT3 Pathway Is Constitutively Activated and Its Inhibition Leads to CLL Cell Death Unaffected by the Protective Bone Marrow Microenvironment

The bone marrow microenvironment promotes proliferation and drug resistance in chronic lymphocytic leukemia (CLL). Although ibrutinib is active in CLL, it is rarely able to clear leukemic cells protected by bone marrow mesenchymal stromal cells (BMSCs) within the marrow niche. We investigated the modulation of JAK2/STAT3 pathway in CLL by BMSCs and its targeting with AG490 (JAK2 inhibitor) or Stattic (STAT3 inhibitor). B cells collected from controls and CLL patients, were treated with medium alone, ibrutinib, JAK/Signal Transducer and Activator of Transcription (STAT) inhibitors, or both drugs, in the presence of absence of BMSCs. JAK2/STAT3 axis was evaluated by western blotting, flow cytometry, and confocal microscopy. We demonstrated that STAT3 was phosphorylated in Tyr705 in the majority of CLL patients at basal condition, and increased following co-cultures with BMSCs or IL-6. Treatment with AG490, but not Stattic, caused STAT3 and Lyn dephosphorylation, through re-activation of SHP-1, and triggered CLL apoptosis even when leukemic cells were cultured on BMSC layers. Moreover, while BMSCs hamper ibrutinib activity, the combination of ibrutinib+JAK/STAT inhibitors increase ibrutinib-mediated leukemic cell death, bypassing the pro-survival stimuli derived from BMSCs. We herein provide evidence that JAK2/STAT3 signaling might play a key role in the regulation of CLL-BMSC interactions and its inhibition enhances ibrutinib, counteracting the bone marrow niche.

PF358 FUNCTIONAL HIERARCHY AMONG PRO-SURVIVAL BCL-2 MEMBERS IN DETERMINING RESISTANCE TO VENETOCLAX IN CLL

Background: The Bcl-2 inhibitor Venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells in peripheral blood (PB) whereas lymph node (LN) responses are less complete. This may be linked to pro-survival signals in the LN, where micro-environmental signals strongly induce Bcl-XL, Mcl-1 and Bfl-1 which are not targeted by Venetoclax. In some patients resistance occurs over time which sometimes can be explained by acquired mutations, but in most cases the mechanism(s) remain unknown. Aims: We hypothesize that changes in expression or shifts in functional interactions among Bcl-2 family members are key to clinical response and refractory disease. Methods: Intracellular FACS staining for Bcl-2, Mcl-1 and Bcl-XL in circulating recent LN emigrants (CD19+/CXCR4high/CD5dim) versus returning (CD19+/CXCR4llow/CD5high) lymphocytes in patient samples treated with Venetoclax. To investigate the functional impact of expression of Bcl-2 family members, profiling with additional BH3 mimetics against Bcl-XL (A1331852) and Mcl-1 (S63845) was applied in a CLL LN model. Primary CLL cells were co-cultured with fibroblasts expressing CD40L in order to mimic T cell help in the LN. Results: Under Venetoclax treatment, levels of Bcl-2 itself and other family members may increase in some patients, early in clinical response. During short-term in vitro incubation with Venetoclax or other BH3 mimetics, we established that Mcl-1 and Bfl-1 can increase but not Bcl-2 or Bcl-XL. The effect on Mcl-1 and Bfl-1 can be explained by a stabilization of these normally short-lived proteins. In vitro CD40 activation renders CLL cells fully resistant to single Venetoclax treatment. Using this model, we find that dual or triple BH3 mimetic combinations targeting Bcl-2, Bcl-XL and Mcl-1 restored killing. Specifically, whereas the IC50 of dual Bcl-2+Mcl-1 targeting is >1uM, combined inhibition of Bcl-2+Bcl-XL has an IC50 around 0.3uM. Bcl-2 and Bcl-XL thus seem to be the most dominant players. However, even when Bcl-2, Bcl-XL and Mcl-1 were inhibited using triple BH3 mimetic treatment, Venetoclax resistance was not fully reversed to unstimulated control (IC50 1 versus 10 nM), highlighting a potential role for Bfl-1. To further dissect the functional roles, Bim co-immunoprecipitation was performed. In PB CLL cells, Bim only binds to Bcl-2 and upon Venetoclax treatment it is released and targets Bax/Bak to kills cells. After CD40 stimulation, Bim shifts predominantly to Bcl-XL and less to Mcl-1. Only when applying triple BH3 mimetic combinations targeting Bcl-2, Bcl-XL and Mcl-1, Bim starts to bind Bfl-1. These observations suggest a hierarchical model in which Bcl-2 is most important and where upon Venetoclax treatment, Bcl-XL and Mcl-1 come into play. When all of these are targeted by triple BH3 mimetics, Bfl-1 is engaged to intercept apoptosis. Summary/Conclusion: We applied BH3 mimetics in primary CLL and showed that combined targeting of Bcl-2 members can abrogate resistance to Venetoclax. Moreover, we dissected the roles of the different Bcl-2 family members and constructed a hierarchical model of Bcl-2 family members, providing new insights into Venetoclax resistance which may have clinical utility.

Pharmacologic inhibition of the ubiquitin-activating enzyme induces ER stress and apoptosis in chronic lymphocytic leukemia and ibrutinib-resistant mantle cell lymphoma cells

With the advent of proteasome inhibitors (bortezomib) and pleiotropic pathway modulators which target cereblon E3 ligase (lenalidomide), the ubiquitin-proteasome system has emerged as a tractable target in non-Hodgkin lymphoma and multiple myeloma. Here we report that TAK-243, a small molecule inhibitor of the ubiquitin-activating enzyme (UAE), induced ER stress and the unfolded protein response in primary chronic lymphocytic leukemia cells, facilitating cell death. Moreover, targeting UAE was effective in ibrutinib-resistant mantle cell lymphoma cell lines and primary cells in vitro. Thus, UAE is a promising target in lymphoid malignancies, including ibrutinib-resistant lymphomas, an area of unmet medical need.

HSP70/HSF1 axis, regulated via a PI3K/AKT pathway, is a druggable target in chronic lymphocytic leukemia.

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.

Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies

Despite the remarkable results of CD19 directed chimeric antigen receptor T cell (CART19) therapy, the durable responses in B cell lymphoma and chronic lymphocytic leukemia (CLL) is limited. This may be due to the loss of CART persistence, poor trafficking and inhibition by the tumor microenvironment. Strategies to enhance CART function are thus needed. Recent data have shown that abnormal expression of receptor tyrosine kinase (RTK) AXL portends poor prognosis. Inhibition of AXL with TP0903, a high affinity AXL inhibitor, has been found to induce robust apoptosis of CLL B cells. We therefore examined the role of AXL inhibition with TP0903 on T cell and CART19 function. First, we investigated the effect of AXL inhibition on naive T cells. Cells stimulated with PMA/Ionomycin and cultured with TP0903 showed significant reduction of Th2 cytokines and inhibitory receptors (Fig 1a). Effector and regulatory T cells (Treg) treated with TP0903 showed a preferential reduction of Tregs (Fig 1b). Next, we investigated the influence of TP0903 on 41BB costimulated CART19. TP0903 led to polarization of CARTs into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP0903 treatment also significantly downregulated inhibitory receptors on activated CARTs, including cytokine release syndrome (CRS) related cytokines (Fig 1c). The combination of CART19 and TP0903 yielded a synergistic cytotoxicity against JeKo in vitro (Fig 1d). We compared the transcriptome of activated CARTs treated with TP0903, and noted >100 genes that were differentially expressed compared to non-treated cells. Among these, cell junction, cell migration, and immune synapse related genes were significantly increased in activated CARTs treated with TP0903. To investigate the effect of AXL inhibition of CARTs with TP0903 in vivo, we established MCL xenografts via the injection of JeKo into NSG mice. The mice were then treated with vehicle alone, TP0903 alone, CART19 alone, or TP0903 + CART19. Three weeks after the treatment, mice were rechallenged with JeKo. Mice treated with CART19 and TP0903 rejected the JeKo challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART persistence (Fig 1e). Finally, we validated our findings in a phase I clinical trial of TP0903 (NCT02729298). T cells isolated from 3 cancer patients showed significant reduction of Tregs. This will be further investigated in a planned phase I clinical trial of TP0903 in relapsed/refractory CLL (NCT03572634). In summary, we showed for the first time that AXL inhibitior polarizes T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes CARTs in B cell malignancies. These findings encourage further study of TP0903 as an enhancer of T cell immunotherapies.

Acitretin Reduces L-Selectin Expression and Tumour Cell Homing in Chronic Lymphocytic Leukaemia (CLL)

Introduction:The microenvironment plays a major role in the survival of Chronic Lymphocytic Leukaemia (CLL) cells by promoting CLL cell growth and drug resistance. The traffic of CLL cells that involve migration from the blood and adhesion to the high endothelial venules (HEVs) facilitates the homing of lymphocytes in the lymph nodes to protect CLL cells and offer them the necessary survival signals. L- Selectin mediates adhesion of CLL cells to LN-microenvironment through a multistep process that entails rolling, sticking, and crawling. Acitretin is a retinoid that used in the treatment of severe psoriasis and less frequently in the control of squamous skin cancer. Acitretin activates nuclear retinoic acid receptors (RAR), resulting in induction of cell differentiation, inhibition of cell proliferation, and inhibition of tissue infiltration by inflammatory cells. It is reported that retinoids induce apoptosis in CLL and improve the sensitivity of CLL cells to fludarabine.Method:We used MEC-1 “CLL like” cell line and CLL cells from known CLL patients to investigate the effect of acitretin on the expression of the major markers that control CLL cells traffic including L-selectin (CD62L), CD49d and CXCR4 (CD184) . We treated CLL cells with increasing doses of acitretin (10 nM up to 10 mM). After treatment for 48 to 72 hours, L-selectin, CD49d and CXCR4 expression was measured using flowcytometry. Dimethyl sulfoxide (DMSO) treated cells were used as a negative control. A Leukocyte adhesion assay was done using CytoSelect Leukocyte-endothelium adhesion assay according to the protocol provided. To assess the mechanism through which L-Selectin reduces the adhesion of CLL cells to the endothelium, we used western blot to measure the effect of acitretin on PI3k-p85.Results:Acitretin in a dose as low as 10 nM significantly reduced the expression of L-selectin in primary CLL cells after treatment for 48 hours (P= 0.0475) and 72 hours (P= 0.0048). On the contrary, acitretin did not change the expression of CD49d or CXCR4. Similar significant reduction in L-selectin expression was recognized when MEC-1 cells were treated with 10 nM of acitretin for 48 hours (P= 0.0179) and 72 hours (P= 0.0159).To test the effect of acitretin on the adhesion of MEC-1 cells to the endothelium; we used CytoSelect adhesion assay that showed significant reduction in the adhesion of MEC-1 cells to human umbilical vein endothelial cells (HUVEC) after treatment with acitretin 10 mM for 48 hours (P= 0.0048). Ibrutinib treatment has also significantly reduced the adhesion of MEC-1 cells to HUVEC cells (P= 0.0045) and when combined with acitretin the reduction in adhesion was more pronounced (P= 0.0003).Finally, to understand the mechanism through which acitretin down regulates the expression of L-selectin and reduces the adhesion of MEC-1 cells, we tested the effect of acitretin on the PI3K pathway using western blot. PI3K is required for the cytoskeleton changes during neutrophil rolling on E‐selectin and we investigated if similar interaction takes place between PI3K and L-selectin in CLL cells. Treatment of MEC-1 cells with acitretin 10 mM for 48 hours resulted in significant down regulation of the PI3K-p85 (P= 0.0007). Treatment with Ibrutinib alone or combined with acitretin did not result in significant change in PI3K- p85. This may explain the mechanism through which acitretin reduces the expression of L-selectin and compromises CLL adhesion to the endothelium.Conclusion:We showed that the retinoid acitretin significantly reduced the expression of L-selectin in CLL cells and MEC-1 “CLL like” cells in vitro. The reduction in L-selectin resulted in reduced adhesion of CLL cells to the endothelium hampering homing of CLL cells to lymph nodes. The mechanism through which acitretin exerts this effect can be explained by down regulation of PI3K-p85. The use of acitretin as an adjunct treatment in CLL can be beneficial, however further research including in vivo studies is required. DisclosuresQuinn:Janssen: Honoraria.

Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies

Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies

NDRG2 mRNA levels and miR-28-5p and miR-650 activity in chronic lymphocytic leukemia

BackgroundNDRG2 is identified as a tumor suppressor gene in many tumors, and functions in cell proliferation, differentiation and apoptosis. Recent data indicate that NDRG2 expression is up-regulated by TP53. Moreover, proposed mechanisms of NDRG2 inactivation include epigenetic silencing of the NDRG2 promoter and down-regulation by microRNAs (miRNAs). However, few studies have ever been done on the role of NDRG2 and the NDRG2-regulating miRNAs interference in chronic lymphocytic leukemia (CLL).MethodsNDRG2 and microRNAs mRNA levels in CLL subjects were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The dual-luciferase reporter assay was performed to determine NDRG2-related miRNAs. Low expression of mature exogenous miRNAs in CLL cells was established by transient transfection. NDRG2 protein levels in CLL cells were detected by western blot. In addition, flow cytometry was conducted to examine the apoptosis of CLL cells.ResultsLower expression of NDRG2 was found in the B-cells from 102 CLL patients compared the 40 normal subjects (P < 0.001). Patients with advanced Binet stage (P = 0.001), high lactate dehydrogenase (LDH) level (P = 0.036), un-mutated immunoglobulin heavy chain variable region gene (IGHV) (P = 0.004) and those with p53 aberrations (P < 0.001) had a markedly lower levels of NDRG2 mRNA. This decrease was associated with briefer time-to-treatment (P = 0.001) and poorer survival (P < 0.001). High expression of miR-28-5p and miR-650 was associated with Binet B/C stage (P = 0.044) and IGHV un-mutated (P = 0.011), as well as Binet B/C stage (P = 0.013) and p53 aberrations (P = 0.037), respectively. Inhibition of miR-28-5p or miR-650 could induce more apoptosis in CLL cells with germline TP53.ConclusionsNDRG2 mRNA levels might be a useful prognostic variable for patients of CLL and up-regulating NDRG2 transcription may be a therapy approach in CLL without p53 aberrations.

Abstract 1881: The microtubule-disrupting drug BNC105 is a potent inducer of apoptosis in AML patient samples

Abstract BNC105 is a phase II potent and highly selective disruptor of tumor microvasculature that causes the rapid onset of hypoxia and necrosis in solid tumors. BNC105 targets the colchicine-binding site on tubulin, causing chronic disruption of adhesion molecules, and was developed to be best-in-class with high specificity to actively dividing cells. It has one of the largest therapeutic windows of its class and has been shown to have direct cytotoxic activity on tumor cells. It is this highly tumor-specific mechanism of action that has positioned BNC105 as a therapeutic with high potential in the hematologic cancer setting. Previous studies of BNC105 have shown that treatment with BNC105 results in the activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and the induction of ATF3 and Noxa, leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. These findings led to the commencement of a phase 1/2 trial of BNC105 in patients with CLL. The present study was designed to investigate the effect of BNC105 treatment on acute myeloid leukemia (AML), a disease that currently has limited treatment options. To assess the utility of BNC105 therapy in this setting, six AML cell lines representing different subtypes, including the high-risk FLT3-ITD subtype, were initially used in proliferation and cytotoxicity assays. The production of reactive oxygen species (ROS), cell cycle distribution and cell signaling by Western blot were all assessed after treatment. All tested AML cell lines were highly sensitive to treatment with BNC105 with an IC50=0.2nM to 1.3nM after 48 hours treatment. Analysis of apoptosis induction revealed cell line-specific effects; however, a consistent dose-dependent increase in phosphorylation of JNK was observed across all cell lines. AML patient samples obtained from the South Australian Cancer Research Biobank (SACRB) were exposed to BNC105 at clinically relevant doses for up to 72 hours and cellular viability and apoptosis induction were assessed by Annexin V/ 7AAD staining and caspase 3 and 7 activation measured. BNC105 induced caspase activity and significantly decreased viability in a dose- and time-dependent manner, including the FLT3 mutant subtype patient samples. In comparison, bone marrow mononuclear cells from healthy controls were much less affected by BNC105. Effects of BNC105 on the leukemic stem cell (LSC) phenotype population were also investigated. The LSC-containing population, measured by CD34/CD38 and GPR56 or CD93 staining, was targeted by BNC105 in all AML patient samples tested. These results suggest that AML cells can be directly targeted by BNC105 at clinically relevant concentrations and hence further clinical investigation of BNC105 is warranted for AML treatment in a patient population with high unmet need. Citation Format: Daniel J. Inglis, Debora A. Casolari, Tran Nguyen, Donna M. Beaumont, Nicole L. Wittwer, David Ross, Richard D'Andrea, Tina C. Lavranos. The microtubule-disrupting drug BNC105 is a potent inducer of apoptosis in AML patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1881.

Circular RNA circ-CBFB promotes proliferation and inhibits apoptosis in chronic lymphocytic leukemia through regulating miR-607/FZD3/Wnt/β-catenin pathway

Circular RNA (circRNA) belongs to the non-coding RNA family and is involved in various human cancers, such as lung cancer and colorectal cancer. Nevertheless, whether circRNA expression is related to chronic lymphocytic leukemia (CLL) progression remains largely unclear. In our study, we investigated the role of circ-CBFB in CLL. We found that circ-CBFB was markedly overexpressed in CLL cells compared to normal controls. Furthermore, we found that circ-CBFB could serve as a diagnostic and prognostic biomarker for CLL patients. We also explored the physiological function of circ-CBFB. We found that circ-CBFB knockdown significantly suppressed CLL cell proliferation, arrested cell cycle progression, and induced cellular apoptosis. In terms of its mechanism, we identified circ-CBFB as a sponge of miR-607, which targeted FZD3. By inhibiting miR-607 availability, circ-CBFB promoted FZD3 expression, leading to the activation of the Wnt/β-catenin pathway and consequent CLL progression. Taken together, our findings revealed that the circ-CBFB/miR-607/FZD3/Wnt/β-catenin regulatory signaling cascade contributes to CLL progression.

A novel ex vivo high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.

Pentoxifylline-Induced Apoptosis in Chronic Lymphocytic Leukemia: New Insights into Molecular Mechanism.

Chronic lymphocytic leukemia (CLL) is an indolent B-lineage neoplasm, characterized by clonal expansion of CD5 positive B cells with constitutive activation of survival pathways including NF-kB. Pentoxifylline, a xanthine-derivative compound indicated for the treatment of microvascular disturbancies, has been suggested to have anti-proliferative and anti-metastatic activities in various types of cancer. In the present study we extend these data showing one of the potential molecular mechanisms through which Pentoxifylline may promote apoptosis in CLL clonal lymphocytes. Peripheral blood mononuclear cells were isolated from 15 CLL patients 0 RAI stage and 15 healthy volunteers and treated for 24 and 48 hours with Pentoxifylline. Apoptosis induction was evaluated through Annexin V and TUNEL assays. Mitochondrial membrane potential depolarization analysis, active Caspase-3 assay, reactive oxygen species generation and Western Blot were assessed to further investigate the alterations induced by Pentoxifylline. We observed a statistically significant occurrence of apoptosis, DNA fragmentation and active Caspase-3 in lymphocytes from CLL patients compared to healthy volunteers after 48 hours of Pentoxifylline treatment. To clarify the molecular mechanism of the drug, we also evaluated the expression levels of NF-kB/p65 and its related proteins. In treated CLL cells, NF-kB/p65 was significantly decreased in comparison to normal cells, whereas we observed a less marked reduction of Bcl-2 expression. The treatment also induced a decrease of Mcl-1 in CLL cells with a greater down-regulation of the anti-apoptotic alternatively spliced isoform. These findings showed that Pentoxifylline induced apoptosis in leukemic cells through a molecular mechanism that involves the NF-kB signaling.

Microenvironmental stromal cells abrogate NF-κB inhibitor-induced apoptosis in chronic lymphocytic leukemia.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is known to play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Several NF-κB inhibitors were shown to successfully induce apoptosis of CLL cells in vitro. Since the microenvironment is known to be crucial for the survival of CLL cells, herein, we tested whether NF-κB inhibition may still induce apoptosis in these leukemic cells in the presence of protective stromal interaction. We used the specific NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). Microenvironmental support was mimicked by co-culturing CLL cells with bone marrow-derived stromal cell lines (HS-5 and M2-10B4). NF-κB inhibition by DHMEQ in CLL cells could be confirmed in both the monoculture and co-culture setting. In line with previous reports, NF-κB inhibition induced apoptosis in the monoculture setting by activating the intrinsic apoptotic pathway resulting in poly (ADP-ribose) polymerase (PARP)-cleavage; however, it was unable to induce apoptosis in leukemic cells co-cultured with stromal cells. Similarly, small interfering ribonucleic acid (siRNA)-mediated RELA downregulation induced apoptosis of CLL cells cultured alone, but not in the presence of supportive stromal cells. B-cell activating factor (BAFF) was identified as a microenvironmental messenger potentially protecting the leukemic cells from NF-κB inhibition-induced apoptosis. Finally, we show improved sensitivity of stroma-supported CLL cells to NF-κB inhibition when combining the NF-κB inhibitor with the SYK inhibitor R406 or the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, agents known to inhibit the stroma-leukemia crosstalk. We conclude that NF-κB inhibitors are not promising as monotherapies in CLL, but may represent attractive therapeutic partners for ibrutinib and R406.

Identification and characterization of a potent and biologically-active PDE4/7 inhibitor via fission yeast-based assays

We previously constructed a collection of fission yeast strains that express various mammalian cyclic nucleotide phosphodiesterases (PDEs) and developed a cell-based high throughput screen (HTS) for small molecule PDE inhibitors. Here we describe a compound, BC54, that is a selective inhibitor of enzymes from the cAMP-specific PDE4 and PDE7 families. Consistent with the biological effect of other PDE4 and PDE7 inhibitors, BC54 displays potent anti-inflammatory properties and is superior to a combination of rolipram (a PDE4 inhibitor) and BRL50481 (a PDE7A inhibitor) for inducing apoptosis in chronic lymphocytic leukemia (CLL) cells. We further exploited PKA-regulated growth phenotypes in fission yeast to isolate two mutant alleles of the human PDE4B2 gene that encode enzymes possessing single amino acid changes that confer partial resistance to BC54. We confirm this resistance to both BC54 and rolipram via yeast-based assays and, for PDE4B2T407A, in vitro enzyme assays. Thus, we are able to use this system for both chemical screens to identify biologically-active PDE inhibitors and molecular genetic studies to characterize the interaction of these molecules with their target enzymes. Based on its potency, selectivity, and effectiveness in cell culture, BC54 should be a useful tool to study biological processes regulated by PDE4 and PDE7 enzymes.

Venetoclax in Patients with Previously Treated Chronic Lymphocytic Leukemia.

Venetoclax is the first BCL2 inhibitor to enter routine clinical practice. It is an orally bioavailable small molecule that binds BCL2 very specifically. Acting as a pharmacologic mimic of the proteins that initiate apoptosis (a so-called BH3 mimetic), venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells, which express high levels of BCL2 and rely on it to maintain their survival. As a single agent, daily venetoclax treatment induced durable responses in 79% of patients with relapsed or refractory CLL or small lymphocytic lymphoma in a phase I study, including complete remissions in 20% of patients. Its use was approved by the FDA in April 2016 for patients with previously treated del(17p) CLL on the basis of a single-arm phase II trial demonstrating a 79% response rate and an estimated 1-year progression-free survival of 72% with 400 mg/day continuous therapy. This review focuses on venetoclax, its mechanism of action, pharmacology, and clinical trial data and seeks to place it in the context of rapid advances in therapy for patients with relapsed CLL, especially those with del(17p) CLL. Clin Cancer Res; 23(16); 4527-33. ©2017 AACR.

Venetoclax for the treatment of patients with chronic lymphocytic leukemia.

Venetoclax is a potent, selective inhibitor of BCL-2, a key regulator of the intrinsic pathway of apoptosis. In preclinical studies, venetoclax bound to BCL-2 with high affinity and rapidly induced apoptosis in chronic lymphocytic leukemia (CLL) cells. In early-phase clinical trials in CLL, venetoclax treatment led to tumor lysis syndrome in some patients with a large tumor burden, but this risk was subsequently mitigated by a revised study design that included lower initial dosing with intrapatient dose ramp up and close tumor lysis syndrome monitoring and prophylaxis. Other toxicities, such as neutropenia and gastrointestinal adverse events, were manageable. Venetoclax monotherapy resulted in durable and deep responses in patients with relapsed, refractory CLL, including for those with deletion 17p, leading to the approval of venetoclax by the US FDA for relapsed or refractory deletion 17p CLL, and recently to additional approvals in Europe and Canada. Trials also suggest that venetoclax induces deeper and more durable responses when used in combination with rituximab, and combination studies with other agents are ongoing. Phase III trials are also underway, and will provide data on the efficacy and safety of venetoclax in combination with monoclonal antibodies and targeted therapies in larger patient populations.

Chrysin as an Anti-Cancer Agent Exerts Selective Toxicity by Directly Inhibiting Mitochondrial Complex II and V in CLL B-lymphocytes

ABSTRACTWe investigated the effect of chrysin on isolated normal and chronic lymphocytic leukemia (CLL) B-lymphocytes and their isolated mitochondria. We report that a selective and significant increase in cytotoxicity, intracellular reactive oxygen species, mitochondrial membrane potential collapse, ADP/ATP ratio, caspase 3 activation and finally apoptosis in chrysin-treated CLL B- lymphocytes. Also we determined that chrysin selectively inhibits complex II and ATPases in cancerous mitochondria. In this study we proved that the ability of chrysin to promote apoptosis in CLL B-lymphocytes performed by selectively targeting of mitochondria. Our findings may provide a potential therapeutic approach for using chrysin to target mitochondria in CLL B-lymphocytes.

HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells in the hematopoietic system and lymphoid tissues. Although inhibitors targeting the B-cell receptor (BCR) pathway have been successful in the treatment of the disease, the underlying mechanisms leading to BCR over-activity in CLL are not fully understood. In this study, we found that HSP90, a highly conserved molecular chaperone, is overexpressed in CLL compared with resting B cells. HSP90 overexpression is accompanied by the overexpression of several BCR kinases including LYN, spleen tyrosine kinase, Bruton tyrosine kinase and AKT. Chemical and immune-precipitation demonstrated that these BCR constituents are present in a multi-client chaperone complex with HSP90. Inhibition of HSP90 with PU-H71 destabilized the BCR kinases and caused apoptosis of CLL cells through the mitochondrial apoptotic pathway. Further, PU-H71 induced apoptosis in the presence of stromal co-culture or cytoprotective survival signals. Finally, genetic knockdown of HSP90 and its client AKT, but not BTK, reduced CLL viability. Overall, our study suggests that the chaperone function of HSP90 contributes to the over-activity of the BCR signaling in CLL and inhibition of HSP90 has the potential to achieve a multi-targeting effect. Thus, HSP90 inhibition may be explored to prevent or overcome drug resistance to single targeting agents.