Spontaneous Apoptosis Research Articles

Abstract 938: Pharmacologic targeting Mcl-1 with AZD5991 induces apoptosis, suppresses mitochondrial respiration and overcomes ibrutinib resistance in non-Hodgkin lymphoma cells

Abstract Bcl-2 family proteins determine cell fate and comprise pro-apoptotic “initiators” (Noxa, Bim, Puma), anti-apoptotic “guardians” (Bcl-2, Mcl-1, Bcl-xL) and pro-apoptotic effectors (Bax/Bak). Venetoclax, a Bcl-2 inhibitor, received regulatory approval in the therapy of chronic lymphocytic leukemia and acute myeloid leukemia. However, venetoclax is modestly effective in non-Hodgkin lymphoma (NHL). Mcl-1 is a short-lived pro-survival protein that is frequently overexpressed in NHL, leading to increased cell survival and drug resistance. Mcl-1 transgenic mice develop aggressive lymphoma. Thus, selective targeting Mcl-1 represents a promising pharmacologic strategy in NHL. AZD5991 is a selective small-molecule inhibitor with high potency against Mcl-1 which has entered clinical trials. Here we explored its pre-clinical activity in NHL. We detected Mcl-1 expression in ten diffuse large B-cell lymphoma (DLBCL) as well as in parental/ibrutinib-resistant JeKo-1 and Mino mantle cell lymphoma (MCL) cell lines. AZD5991 suppressed proliferation and induced apoptosis in DLBCL cells in a dose-dependent manner, as well as in parental/ibrutinib-resistant MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction, including mitochondrial membrane depolarization, decreased mitochondrial mass, increased mitophagy, and increased levels of reactive oxygen species in both DLBCL and parental/ibrutinib-resistant MCL cells. Mcl-1 inhibition downmodulated basal and maximal respiratory capacity in sensitive, but not in resistant cells, as determined by the Seahorse assay, further indicating mitochondria compromise as a mechanism of susceptibility to Mcl-1 inhibition. An inhibitors screening assay identifies that AZD5991 demonstrated synergy with other BH3-mimetics. Co-treatment of DLBCL cells with Bcl-2/xL inhibitors AZD4320, venetoclax and navitoclax overcame resistance to AZD5991. Finally, Primary MCL cells were co-cultured with control, CD40L- or BAFF-expressing stroma which partially mimics the lymph node microenvironment and induces resistance to spontaneous and drug-induced apoptosis. Mcl-1 inhibition induced apoptosis of primary MCL cells in both control and BAFF-expressing conditions, accompanied by decreased mitochondrial respiration. Meanwhile, CD40L stroma rendered protection from AZD5991 which was partially overcome by co-treatment with venetoclax. In summary, Mcl-1 inhibition using selective BH3-mimetic AZD5991 restricts cell proliferation and induces apoptosis in a subset of DLBCL, parental/ibrutinib-resistant MCL cells and primary neoplastic B cells. Mcl-1 inhibition leads to mitochondrial dysfunction and inhibition of cellular respiration. Resistance to Mcl-1 inhibition may be overcome by concurrent targeting of alternative anti-apoptotic proteins (Bcl-2/xL) in NHL. Citation Format: Tingting Liu, Vi Lam, Elana Thieme, Xiaoguang Wang, Fei Xu, Steve Kurtz, Jeff Tyner, Olga V. Danilova, Alexey Danilov. Pharmacologic targeting Mcl-1 with AZD5991 induces apoptosis, suppresses mitochondrial respiration and overcomes ibrutinib resistance in non-Hodgkin lymphoma 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 938.

Abstract 2990: In vitro drug sensitivity screening platform for primary chronic lymphocytic leukemia (CLL) patient samples

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. It is characterized by proliferation and accumulation of monoclonal, small, mature CD5+ B-cells in peripheral blood, bone marrow and secondary lymphoid organs. The current treatment regimens have improved overall survival but CLL patients will eventually experience relapse. The disease is still incurable making it a necessity to discover and screen for new drugs. Patient derived CLL specimens undergo spontaneous apoptosis when grown in culture and is a major limitation to screening for new therapeutic drugs. We have established an in vitro culture conditions which supports growth and proliferation of patient derived primary CLL cells. Champions has a bank of primary CLL patient samples across different Rai stage, genetic mutations and relapsed and refractory cases. We tested nine CLL samples (models) for proliferation. All nine models proliferated 1.5 to 9.6-fold in the optimized media in 6 days, as measured using CellTiter-Glo. Flow cytometric analysis showed that the CLL cells mostly retained the primary phenotypic characteristic CD5+CD10-Cd19+CD20+ even after being in the culture for 6 days. Next, we screened for sensitivity of primary CLL patient samples (N=9) against known standard of care (SOC) drugs venetoclax, ibrutinib, idelalisib, chlorambucil and cytarabine. Primary PBMC patient samples were seeded in 96 well plates in the enriched media and treated with respective drugs over a concentration range over 5 logs. Drug sensitivity was assessed using CellTiter-Glo® luminescent cell viability assay on day 6. Our in vitro assay indicated varied sensitivity of patient samples to approved standard of care drugs. A relapsed bendamustine pre-treated patient sample was sensitive to all the SOC drugs tested. In addition to drug response, whole exome sequencing and RNAseq are being conducted on these samples, to compare mutational analyses with drug responsiveness. With clinically annotated patient-derived CLL samples, WES and RNAseq plus drug response to standard of care provides a comprehensive ex vivo platform for the preclinical testing of drug candidates, which may not only provide information on agent efficacy, but that can permit potential biomarker mining and exploration of patient selection criteria for investigational new agents in CLL. Citation Format: Bandana Ajay Vishwakarma, Amy Wesa. In vitro drug sensitivity screening platform for primary chronic lymphocytic leukemia (CLL) patient samples [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 2990.

Atovaquone Demonstrates Single Agent Activity and an Additive Effect in Combination with Cytotoxic Chemotherapy (Cytarabine and Daunorubicin) in AML

Background: To improve outcomes for pediatric acute myeloid leukemia (AML), identifying and targeting mechanisms of therapy resistance is critical. Recently, the antimicrobial drug atovaquone (AQ) was shown to reduce IL-6/STAT3 signaling in vitro and to improve relapse-free survival in adult AML patients who used AQ for prophylaxis after bone marrow transplant (Xiang et al, Blood, 2016). Clinically, this FDA approved drug is well tolerated, even in patients with organ dysfunction. It has established pediatric dosing, and plasma concentrations at standard doses are typically 40-80µM. We performed in vitro and in vivo experiments to evaluate the efficacy of AQ as a single agent and in combination with standard induction chemotherapy (cytarabine, daunorubicin, and etoposide). In order to ensure clinical relevance, all experiments examined cytotoxicity at clinically achievable doses.Methods: AML cell lines were incubated with vehicle control or increasing doses of AQ (1µM - 30µM) for 24, 48, 72, or 96 hours, and then apoptosis was quantified by Annexin-V staining. Primary pediatric AML cells were exposed to 30µM AQ for the same time periods. To reduce spontaneous apoptosis of primary AML cells over prolonged culture, samples were cultured in HS5 stromal cell-conditioned media (CM) or co-cultured with HS5 stromal cells. For in vivo studies, female NSG mice were injected with 5x105 THP-1 cells expressing firefly luciferase (THP-1.ffluc). Mice were randomly assigned to receive AQ 200 mg/kg daily (n=6) or vehicle (n=5) by oral gavage, starting on day 1, and disease burden was monitored by bioluminescent imaging 3 times a week. A cohort of male mice receiving VC or AQ starting the day of injection with THP-1.ffluc cells were followed for survival (n=7 for each group). For in vitro drug combination cytotoxicity assays, 103 THP-1.ffluc cells per well were plated in 96 well plates with media only, single drugs at increasing concentrations, and AQ+chemotherapy combinations (AQ 1-15uM, AraC 0.1-3uM, Dauno 0.03-0.6uM, VP-16 0.3-3uM). After 48 hours, viability was determined by luminescence. Background luminescence of media-only wells was subtracted from all values and means of 4 replicates were used for analysis. All values were normalized to the cells + vehicle wells, which were set to 0 to quantify the percent affected. The Calcusyn method was used to calculate the combination index (CI). An AQ+chemotherapy combination was considered synergistic if CI<0.8, additive if CI>0.8 and <1.2, or antagonistic if CI>1.2.Results: All 7 AML cell lines demonstrated time and dose-dependent induction of apoptosis in response to 1-30µM doses of AQ. IC50s were within the range of typical clinically achievable doses. For AML cell lines, the IC50s (in µM) after 72 hour incubation were: MOLM13 13.7, MV4-11 14.0, THP-1 15.7, NB4 16.9, Kasumi 1 18.6, HL60 33.7, KG1 98.9. AQ was generally less potent against the CML cell line K562 and the precursor B ALL cell line RS4 (72 hour IC50s were 116.6 and 81.4 respectively). All tested primary samples demonstrated AQ induced apoptosis that increased over time. After subtracting spontaneous apoptosis at each timepoint, the apoptosis induced by AQ at 72 hours for 5 patient samples supported by co-culture were 36, 37, 39, 67 and 87%. Annexin-V+% for the same patient samples supported by CM were 19, 13, 51, 47, and 24%. Bioluminescent imaging of female NSG mice xenografted with THP-1.ffluc cells and treated with single agent AQ daily starting on the day of cell injection demonstrated decreased disease burden compared to control mice (for example on day 21, the average radiance of VC treated mice vs AQ treated mice was 2 x 105 vs 6.3 x 104 p/s/cm²/sr; p=.0048 by ANOVA). Single agent AQ prolonged survival of male xenografted mice with all VC mice succumbing to disease at a median of 63 days vs 1/7 AQ treated mice by day 70 (p=.003 by log-rank test). Our drug combination assays found an antagonistic effect in combination with etoposide, and an additive effect with cytarabine and daunorubicin.Conclusions: Atovaquone induced dramatic levels of apoptosis of AML cells at clinically achievable doses in vitro ; reduced disease burden and improved survival in AML xenografts; and had an additive effect with standard chemotherapy drugs for pediatric AML. Our data support further evaluation of atovaquone for pediatric AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Primary Chronic Lymphocytic Leukemia Cells Can be Maintained Long-Term in Serum-Free, Cytokine-Free 3D Culture

Chronic Lymphocytic Leukemia (CLL) cells undergo rapid and spontaneous apoptosis when cultured in vitro, challenging the study of disease biology. Conventional CLL culture platforms are dependent on addition of high concentrations of soluble molecules, animal- derived serum, adhesion proteins or allogeneic/xenogeneic stromal cells. We have hypothesized that a three-dimensional (3D) architecture may create a niche-like culture system, enabling CLL cell maintenance within a self-perpetuating microenvironment, in the absence of additional growth factors.In order to assess optimal culture parameters, several conditions were tested: (1) Initial seeding densities (2, 10 and 40 x 106 cells/scaffold); (2) Culture media (IMDM or RPMI + 10% FBS, StemSpan SFEM and Hybridoma SFM); (3) Oxygen tension (21% or 5% O2) and (4) Scaffold coating (uncoated, collagen, RGD). Primary mononuclear cells were seeded onto polyurethane scaffolds and complete media exchanges were performed every third day. The system has enabled successful culture for up to 54 days in 6 out of 7 primary patient samples, 5 of them isolated from peripheral blood (PB) and 1 from bone marrow (BM). Conventional cultures, set up in parallel, died out by day 7.On days 1, 7, 14 and 28, cultures were evaluated for viable cell expansion in the scaffold with Cell Titer Glo 3D (CTG), for morphological characteristics of CLL lymphocytes by May-Grunwald Giemsa (MGG) staining and for in situ niche-like structure formation with scanning electron microscopy (SEM) and fluorescence confocal microscopy. In both serum-containing media (RPMI and IMDM), cultures established with 2 x 106 cells/scaffold showed a reduction in CTG at day 28 compared with that at day 1 (p<0.01, N=2, n=2), whereas higher seeding densities maintained ATP content and metabolism in situ, suggesting improved maintenance of viable cells in scaffolds. For higher seeding densities, SEM images of scaffolds at day 28 show cells clustering in niches and MGG-stained cells show morphological characteristics of CLL, similar to those isolated from patients at day 0. Cultures with 1 x 107 cells/scaffold in hypoxia (5% O2) and in StemSpan SFEM also showed reduction in CTG from days 1 to 28 (p<0.01, N=3, n=2), suggesting lack of in situ proliferation in these conditions. Furthermore, functionalization of scaffolds with collagen or RGD coating did not improve culture dynamics, displaying no difference between conditions at days 1 or 28 with CTG and created more debris and cellular anomalies when visualised with MGG. In contrast, CLL cells morphologically identical to those isolated from the patient were retrieved from the uncoated scaffolds.When comparing RPMI + 10% FBS (FBS) and serum-free media Hybridoma SFM (SF), primary CLL cells were seeded at 1 x 107 cells/scaffold. Scaffold-extracted and supernatant cells were counted and analysed for viability with Guava Via Count assay and assessed for morphology with MGG at every medium exchange. At days 1, 14 and 28 of culture, cells extracted from scaffolds were also analysed with propidium iodide and Annexin V staining for detection of early apoptosis. In the first 8 days of culture, 10.2% of seeded cells egressed from FBS culture, compared with 5.9% in SF, both stabilizing thereafter with similar numbers of viable cells migrating into the supernatant. After 8 days, cells were more viable in SF than FBS in the supernatant (43.3% and 21.8%, respectively; p=0.0097) as well as when cells extracted from scaffolds on days 14 (61.9% and 32.6%, respectively; p<0.0001) and 28 (61% and 16%, respectively; p=0.0013). Consistent with these results, in situ proliferation was higher for SF than FBS (n=3, p<0.05). Using confocal microscopy, DAPI, and an amine reactive dye, widespread in situ scaffolddistribution of cells was observed in SF by day 28 with formation of cellular clusters inside the scaffold. Furthermore, lymphocytes and nurse-like cells (vimentin positive) were detected at day 28 in SF by confocal microscopy in niche-like structures.To the best of our knowledge, this work constitutes the first long-term culture (up to 8 weeks) of patient-derived primary CLL cells without cytokines, serum or feeder layers by using a 3D scaffold and high cell density. This platform may address an unmet need for a culture system which could represent the native disease environment, and a potential drug-testing platform. DisclosuresNo relevant conflicts of interest to declare.

Phase II/1b Trial of Ibrutinib Plus Prednisone and Rituximab Induction Followed By Venetoclax Plus Ibrutinib Maintenance in Newly Diagnosed High-Risk Chronic Lymphocytic Leukemia

Introduction: Therapy options for chronic lymphocytic leukemia (CLL) are rapidly changing with the approval of ibrutinib, an irreversible Bruton's tyrosine kinase inhibitor, and venetoclax, a Bcl2 inhibitor. In the RESONATE studies, ibrutinib showed significant improvements in overall response rates (ORR), progression-free-survival (PFS), and overall survival (OS). Nonetheless, complete response (CR) rates were only about 4%.During the first few weeks of treatment, ibrutinib will mobilize CLL cells from tissue sites into peripheral blood. Rituximab, a chimeric monoclonal CD20 antibody, when used with ibrutinib, has shown to immediately clear CLL from the blood with durable responses but with CR's of only 8%.Venetoclax is approved for relapsed or refractory CLL with 17p deletion. In a phase 2 study, at a median follow-up of 12.1 months, the ORR was ~79%, but with only 16% CR's. Amongst the 45 patients of whom minimal residual disease (MRD) was assessed, 24 were negative for MRD.In vitro studies have shown that nurse-like cells (NLCs) will protect CLL cells against spontaneous apoptosis by producing chemokines and interleukins such as SDF1, suggestive of a mechanism of ibrutinib resistance. SDF1 was shown to bind to its receptor CXCR4 located on CLL cells and activate a signal transduction pathway that would reduce spontaneous apoptosis. Dexamethasone was found to decrease NLC viability. This suggests that a combined therapy of ibrutinib plus steroids along with rituximab and venetoclax may be necessary to achieve deeper CR's in high-risk CLL.In addition, CLL with overexpression of exosome miR-155 is associated with a poor response to chemoimmunotherapy. In prior preclinical studies, exosome miR-150 and miR-155 have been shown to increase with BCR activation via α-IgM stimulation. We hope that via next generation microRNA sequencing from our patients' CLL plasma cells we will observe a decrease in the levels of exosome miR-150 and miR-155 in response to inhibition of BCR-pathway by ibrutinib as seen in preclinical studies.Methods: Patients with newly diagnosed high-risk CLL per FISH studies will be enrolled. Ibrutinib will be administered orally 420 mg daily, rituximab will be dosed at 375 mg/m2 intravenously on days 1, 8, 15, 22 of cycle 1 and on day 1 for cycle 2-6. Prednisone will be administered as 10 mg one week on, one week off per cycle. Each cycle will last 28 days. After 6 cycles, if patients have MRD, venetoclax will be added in on a weekly schedule with weekly ramp up from 20mg to 50mg, 100mg, and 200mg for a total duration of 6 months plus ibrutinib at 420mg as maintenance until disease progression or drug intolerance. There is the option to reintroduce venetoclax, prednisone, or rituximab if patients progress during maintenance.Results: The primary objectives of this study are to evaluate safety and MRD negative CR rates based on IWG-CLL criteria. For the evaluation of safety, the proportions of the 29 subjects with any adverse events, each specific adverse event, and study-related adverse events will be reported along with the 95% confidence intervals. The proportion of the 29 subjects who demonstrate a MRD negative CR will be reported along with the 95% confidence interval. A sample size of 29 will produce a two-sided 95% confidence interval with a width≤ 0.380.In addition, Kaplan-Meier method will be performed to evaluate time to first MRD negativity (TTMN), time to first relapse (TTFR), PFS and OS, respectively. Based on the Kaplan-Meier curve, the median time to TTMN, TTFR, PFS and OS, respectively, will be reported along with the 95% confidence interval if it exists. Correlation coefficients will be derived to describe the relationship between continuous endpoints and Kendall's tau will be derived to describe the relationship between categorical endpoints.There is no planned interim analysis for the CR rate. However, at any time point if an excessive number of adverse events are observed, the study will be terminated.Conclusion: In this Phase IB/II clinical trial, we seek to observe prolonged response rates of ibrutinib via inhibiting NLC's by adding prednisone. Also, we anticipate that rituximab will further amplify the CR's. For maintenance therapy, we hope that by combining venetoclax with ibrutinib in high-risk CLL patients who were unable to achieve negative MRD's, we will be able to induce deeper remissions. [Display omitted] DisclosuresPersky:Genentech: Consultancy; MorphoSys: Other: Independent Data Monitoring Committee member ; Verastem: Consultancy; Spectrum Pharmaceuticals: Research Funding.

TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in Diffuse Large B-Cell Lymphoma (DLBCL) Cells

Despite the promise of targeting B-cell receptor-associated signaling kinases in indolent lymphomas, new agents in this class show limited activity in DLBCL, which remains an unmet clinical need. Ubiquitin-proteasome systems (UPS) are altered in cancer, leading to destabilization of tumor suppressors (e.g., TP53), overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. We and others have shown that neoplastic B-cells exhibit a state of heightened cellular stress and thereby are susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Proteasome inhibitors (e.g., bortezomib) have demonstrated clinical activity in B-cell neoplasia. Our group has previously targeted proximal components of the UPS in B-cell neoplasia by disrupting the Nedd8 pathway with pevonedistat, which inhibits the function of a subset of E3 ubiquitin ligases known as cullin-ring ligases. Here we investigated an alternative approach to targeting the UPS in DLBCL using TAK-243, a first-in-class small molecule UAE inhibitor. By inhibiting UAE, TAK-243 blocks ubiquitin conjugation, disrupting monoubiquitin signaling as well as global protein ubiquitination. Here we demonstrate that UAE inhibition effectively kills DLBCL cells via induction of ER stress.Experiments were conducted in activated B-cell (ABC)-type (OCI-LY3, U2932) and germinal center (GC)-type (OCI-LY19, SUDHL4/10/16, VAL) DLBCL cell lines, and primary neoplastic B-cells derived from peripheral blood and lymph nodes of patients with CLL and DLBCL, respectively. CD40L-expressing stromal cells were used to induce NFκB activation in primary cells. TAK-243 was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA).TAK-243 induced ER stress and the UPR , followed by rapid apoptosis, as evidenced by PARP cleavage within 2 h, in both DLBCL cell lines and primary neoplastic B-cells. 24 h drug treatment yielded an IC50 of ~50 nM, as estimated by Annexin V expression. TAK-243 induced apoptosis of DLBCL cell lines independent of cell-of-origin (ABC- vs. GC). By contrast, primary T cells were significantly less sensitive to TAK-243.Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and subsequent degradation of proteins controlled by the UPS in neoplastic B-cells. TAK-243-induced UPR occurred within 2 h, as evidenced by oligomerization and autophosphorylation of PERK, induction of BiP/GRP78 and activation of eIF2α. After 4 h of treatment, cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34 (10-fold) and NOXA (4-fold), accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of MCL-1, activation of BAK, and ultimately cleavage of caspase-3 and PARP.TAK-243 inhibited both canonical and non-canonical NFκB pathways in neoplastic B-cells, as evidenced by accumulation of IκBα, a negative pathway regulator, and diminished p100/p52 processing, respectively. We and others have shown that CD40L induces NFκB and promotes resistance to spontaneous and drug-induced apoptosis in primary neoplastic B-cells. By contrast, CD40L-stimulated cells were fully sensitive to TAK-243 (IC50=50 nM). Furthermore, TAK-243 showed lower IC50 compared to bortezomib in DLBCL cell lines and primary samples, and induced accelerated and more pronounced ER stress and NFκB inhibition.We observed increased MYC mRNA transcript and protein expression in neoplastic B-cells following treatment with TAK-243. DLBCL cells expressing high baseline MYC levels (by protein expression) showed enhanced susceptibility to TAK-243-mediated apoptosis compared with MYC-low cells. Engineered expression of MYC sensitized OCI-LY3 cells to TAK-243, while siRNA-mediated downregulation of MYC resulted in decreased apoptosis. It has been suggested that MYC may sensitize cells to apoptosis through the extrinsic pathway. Indeed, we found that TRAIL ligand sensitized DLBCL cell lines to TAK-243.Thus, the UAE inhibitor TAK-243 induces ER stress, abrogates NFκB pathway activation and promotes apoptosis in DLBCL cell lines and primary neoplastic B-cells. Its in vitro activity compares favorably with bortezomib. Targeting UAE is a novel approach to disrupt the ubiquitin-proteasome system which may hold promise in therapy of B-cell malignancies. DisclosuresBerger:Takeda Pharmaceuticals International Co.: Employment.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Susceptibility to Spontaneous Apoptosis of Peripheral Blood Granulocytes from Patients with Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia.

Introduction: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemias (CMML) constitute a heterogeneous group of clonal hematopoietic diseases. Cytopenias are induced by an apoptosis-mediated bone marrow suppression mechanism. Survival blood cells may exhibit resistance to bone marrow apoptosis and may constitute useful markers of the disease.Patients and Methods: Peripheral blood granulocytes were isolated from 14 healthy volunteers, 10 patients with MDS (AR 5, ARSA 1, AREB 4) and 4 patients with CMML using a standard method with dextran sulfate and ficoll-hypaque. Apoptosis was quantified by the percentage of anexina V (ANV) and propidium iodide (PI) cells, changes in mitochondrial membrane potential (JC-1) using flow cytometry and expression of bcl-2, bax, bcl-x, XIAP and mcl-1 by Western blot analysis at 0h. Granulocyte apoptosis was evaluated at 0h and after incubation at 37°C for 24h in RPMI medium enriched with serum from each individual. Statistical significance was determined using the two-tailed Student's t-test considering p<0.05 significant values.Results: Elevated percentages of early (ANV+ IP-) and late (ANV+ IP+) apoptotic granulocytes were found after incubation for 24h in patients and volunteers. Differences in the ratio of early and late apoptotic granulocytes between patients and volunteers were not statistically significant. Similar results were obtained when changes in mitochondrial membrane potential were analyzed. Circulating granulocytes from healthy volunteers and from patients contained high levels of proapoptotic Bax protein. No patient expressed antiapoptotic bcl-2, bcl-x, XIAP and mcl-1 proteins.Conclusion: Granulocytes from patients with MDS and CMML do not exhibit lower susceptibility to spontaneous apoptosis as compared to healthy individuals.

Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia

Hypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

TBC1D8B, a GTPase-activating protein, is a novel apoptosis inducer.

Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.

EMBR-15. PRC2 COMPLEX ENFORCES NEURONAL LINEAGE AND SUPPRESSES TUMOR GROWTH IN SHH MEDULLOBLASTOMA

Hyperactivation of Sonic Hedgehog (SHH) signaling pathway drives tumor progression in the largest medulloblastoma subgroup. During cerebellar development, promoters of SHH target genes show inhibitory trimethylation of histone H3 at lysine 27 (H3K27me3), mediated by the Polycomb Repressive Complex 2 (PRC2). Here, we explored the regulation of cerebellar growth and medulloblastoma tumorigenesis by PRC2 complex components EED and EZH2. For developmental studies, we conditionally deleted Eed or Ezh2 in the Atoh1 lineage that gives rise to the cerebellar granule neuron progenitors (CGNP) that are cells of origin for SHH medulloblastomas. For tumor studies, we bred the conditional Eed- or Ezh2-deleted mouse lines with mice genetically engineered to develop SHH medulloblastoma. Our developmental studies showed that Eed was absolutely required for cerebellar growth. Eed-deleted CGNPs underwent aberrant, myocyte-like differentiation and spontaneous apoptosis, resulting in cerebellar hypoplasia. In contrast, Ezh2 deletion produced no developmental phenotype, despite blocking all H3K27me3 in CGNPs. Our tumor studies showed that Eed-deleted medulloblastomas similarly showed aberrant, myocyte differentiation, but unlike CGNPs, did not undergo widespread apoptosis. Eed-deleted medulloblastomas progressed more rapidly than control tumors, indicating that the inappropriate, muscle-like differentiation did not slow tumor growth. Ezh2-deleted medulloblastomas similarly progressed more rapidly than controls. Our data show that the PRC2 complex acts to enforce neuronal lineage commitment in both development and tumorigenesis and to restrain tumor growth in SHH medulloblastoma. Myocyte differentiation in Eed-deleted tumors suggests that PRC2 loss of function may contribute to the medullomyoblastomas that have been observed in patients. The differences in developmental phenotype show that EZH2 and EED functions are non-identical and can be dissociated, while similar increase in tumor progression show tumor suppressive functions for both EED and EZH2.

A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.

GFI-1 overexpression promotes cell proliferation and apoptosis resistance in mycosis fungoides by repressing Bax and P21.

Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma. The majority of patients with advanced stage MF are resistant to conventional chemotherapy and thus have a poor prognosis. The transcriptional repressor growth factor independence-1 (GFI-1) serves an important role in the development of T-cells. The results of the present study demonstrated that the expression of GFI-1 at different clinical stages of MF was significantly higher compared with benign inflammatory dermatoses, and there was a significant association with disease progression. Gene knockdown of GFI-1 results in the inhibition of Hut-78 cell proliferation and clone formation in vitro, cell cycle arrest and spontaneous apoptosis, upregulation of cell cycle-related P21, as well as the apoptosis-related proteins Bax and Caspase-3, and downregulation of CDK2. Using luciferase assays, and mutational analysis, it was demonstrated that GFI-1 directly regulated the transcription of P21. The results of the present study highlighted a potential molecular therapeutic approach for the treatment of advanced MF.

Multiple cell death pathways triggered by temperature-mediated synergistic effect derived from chiral phototheranostic ablation nanoagents

Inorganic nanomaterials equipped with photosensitive chirality are well-expected as panaceas for many biological issues such as Parkinson's disease, DNA cleavage and cancer therapy owing to their versatile physical and chemical properties. Herein, we employ small size chiral MoO3-x nanodots capped with amino acid (cysteine) as chirality-dependent contrast agents/ photosensitizers for in vivo photoacoustic imaging guided photothermal therapy. Due to the enhanced incident-light-selective absorption at near-infrared (NIR) region and rapid body clearance capability, chiral MoO3-x show complete tumor ablation within 10 days without apparent in vivo toxicity when enantiomerically irradiated with corresponding circularly polarized NIR laser. More importantly, due to the mild temperature photothermal treatment, such chiral nanosystems exhibit a delayed cell apoptosis effect which is confirmed by the apoptosis related gene expression and immunofluorescence analysis in both intrinsic and extrinsic pathways unveiling new insights on synergistic relations between photothermal effects and gene expressions. This concept of inorganic theranostic nanoagent qualified with short photo-irradiation time for photothermal therapy and spontaneous cell apoptosis provides an efficient and biologically safe cancer treatment approach which integrates the advances of chiroptics and photo-gene related therapy strategies.

STAT3 induces the expression of GLI1 in chronic lymphocytic leukemia cells

The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1 activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1 and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1 as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.

MCPIP-1 Restricts Inflammation via Promoting Apoptosis of Neutrophils.

Monocyte chemoattractant protein-induced protein-1 (MCPIP-1) is a potent inhibitor of inflammatory response to pathogens. Acting as endonuclease against transcripts of inflammatory cytokines or transcription factors MCPIP-1 can significantly reduce the cytokine storm, thus limiting the tissue damage. As the adequate resolution of inflammation depends also on the efficient clearance of accumulated neutrophils, we focused on the role of MCPIP-1 in apoptosis and retention of neutrophils. We used peritoneal neutrophils from cell-specific MCPIP-1 knockout mice and showed prolonged survival of these cells. Moreover, we confirmed that MCPIP-1-dependent degradation of transcripts of antiapoptotic genes, including BCL3, BCL2A1, BCL2L1, and for the first time MCL-1, serves as an early event in spontaneous apoptosis of primary neutrophils. Additionally, we identified previously unknown miRNAs as potential binding partners to the MCPIP-1 transcript and their regulation suggest a role in MCPIP-1 half-life and translation. These phenomena may play a role as a molecular switch that balances the MCPIP-1-dependent apoptosis. Besides that, we determined these particular miRNAs as integral components of the GM-CSF-MCPIP-1 axis. Taken together, we identified the novel anti-inflammatory role of MCPIP-1 as a regulator of accumulation and survival of neutrophils that simultaneously promotes an adequate resolution of inflammation.

Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions

Death-fold domains constitute an evolutionarily conserved superfamily that mediates apoptotic signaling. These motifs, including CARD (caspase recruitment domain), DD (death domain), and DED (death effector domain), are believed to exert their effects solely through homotypic interactions. Herein we demonstrate that the CARD-containing protein ARC engages in nontraditional death-fold interactions to suppress both extrinsic and intrinsic death pathways. The extrinsic pathway is disrupted by heterotypic interactions between ARC's CARD and the DDs of Fas and FADD, which inhibit Fas-FADD binding and assembly of the death-inducing signaling complex (DISC). The intrinsic pathway is antagonized by ARC-Bax binding, involving ARC's CARD and the Bax C terminus. This inhibits Bax activation and translocation to the mitochondria. Knockdown of endogenous ARC facilitates DISC assembly and triggers spontaneous Bax activation and apoptosis. Conversely, physiological levels of ARC suppress these events. These studies establish a critical role for nonhomotypic death-fold interactions in the regulation of apoptosis.

EIF3B promotes cancer progression in pancreatic cancer

Background This study aimed to analyze the relative expression of Eukaryotic Translation Initiation Factor 3 Subunit B (EIF3B) in pancreatic cancer and elucidate its contribution to this disease. Methods Relative expression of EIF3B in pancreatic cancer was analyzed by immunohistochemistry. Cell viability was determined by the MTT assay and cell proliferation was measured by direct cell counting. Cell apoptosis was detected by Annexin V staining followed by flow cytometry analysis, and cell cycle was analyzed by PI staining. The differential expression gene analysis was performed by microarray. Tumor progression in response to EIF3B deficiency in vivo was investigated using the xenograft tumor model. Results We found aberrantly high expression of EIF3B in pancreatic cancer, which associated with unfavorable prognosis. Knockdown of EIF3B greatly compromised cell viability and proliferation in both SW1990 and PANC-1 cells. Furthermore, EIF3B deficiency induced cell cycle arrest and spontaneous apoptosis. In vivo tumor progression was significantly suppressed by EIF3B silencing in the xenograft mouse model. Mechanistically, we characterized down-regulation of CDH1 and IRS1 and up-regulation of DDIT3, PTEN and CDKN1B, in response to EIF3B knockdown, which might mediate the oncogenic effect of EIF3B in pancreatic cancer. Conclusions Our data uncovered the oncogenic role of EIF3B in pancreatic cancer.

Role of DACH1 on Proliferation, Invasion, and Apoptosis in Human Lung Adenocarcinoma Cells.

To investigate DACH1 protein expression in lung cancer tissue and matched paracancerous tissue and explore its effect on proliferation, invasion, and apoptosis in human lung adenocarcinoma cells (HLACs). Tumor tissue and matched paracancerous tissue were collected from 46 patients with pathologically diagnosed lung cancer. RT-PCR was performed to detect DACH1 mRNA expression and immunohistochemistry to measured DACH1 protein expression. To determine the effect of DACH1 on lung cancer behavior, small interfering RNA (siRNA) was used to silence DACH1 expression in A549 cells. The impact on the proliferation of tumor cells was then observed by MTT assay, changes in the invasion of tumor cells were identified using transwell chamber assay, and the effects on apoptosis in the cell line were detected using flow cytometry. The expression of DACH1 mRNA and DACH1 protein was significantly decreased in lung cancer tissue versus matched paracancerous control tissue. The silencing of DACH1 expression in A549 cells significantly enhanced cell proliferation, significantly increased cell invasion, and significantly reduced spontaneous apoptosis. DACH1 is downregulated in lung adenocarcinoma tissue. In vitro assessment shows that DACH1 functions as a tumor suppressor, suggesting its potential use as a new target for lung cancer treatment.

Влияние хлорида лития на активацию нейтрофилов при развитии синдрома системного воспалительного ответа у пациентов после операций на сердце с использованием аппарата искусственного кровообращения

Цель исследования - изучение in vitro действия хлорида лития на активность нейтрофилов человека при действии сывороток пациентов с синдромом системного воспалительного ответа, развившемся после операций на сердце с искусственным кровообращением. Методика. Исследование проводили in vitro на нейтрофилах, выделенных из крови 6 здоровых доноров. Нейтрофилы активировали при помощи сыворотки пациентов с синдромом системного воспалительного ответа (ССВО), перенесших операции на сердце с искусственным кровообращением (ИК). Активность нейтрофилов оценивали с использованием флуоресцентных антител к маркерам дегрануляции CD11b и CD66b. Уровень апоптоза и некроза нейтрофилов оценивали через 22 ч после выделения из крови здоровых доноров; количественная оценка была проведена с использованием аннексина V и иодистого пропидия на проточном цитофлуориметре. Интактные и активированные нейтрофилы обрабатывали раствором хлорида лития в концентрациях 0,3; 3,0 и 9,0 мМ. Результаты. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО после операций на сердце с ИК увеличивала экспрессию CD11b в 1,5 раза и экспрессию CD66b в 1,4 раза в сравнении с экспрессией на интактных нейтрофилах. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО и раствором хлорида лития в концентрациях 3,0 и 9,0 мМ приводило к статистически значимому снижению уровня экспрессии CD11b CD66b на поверхности нейтрофилов в сравнении с активированными контрольными. Установлено, что хлорид лития в концентрациях 3,0 и 9,0 мМ возвращал уровни экспрессии CD11b и CD66b на активированных нейтрофилах к уровню экспрессии на интактных нейтрофилах. В концентрации 0,3 мМ хлорид лития, используемый при инкубации с активированными нейтрофилами, не вызывал значимого снижения экспрессии CD11b и CD66b относительно контрольных активированных нейтрофилов. Экспрессия CD11b и CD66b на активированных нейтрофилах при их инкубации с хлоридом лития в концентрации 0,3 мМ была значимо выше относительно экспрессии данных молекул на интактных нейтрофилах. Сыворотка пациентов с развившемся ССВО снижала спонтанный апоптоз нейтрофилов, а раствор хлорида лития в концентрации 3,0 или 9,0 мМ, добавленный в среду инкубации, увеличивал способность нейтрофилов к спонтанному апоптозу. Заключение. Хлорид лития оказывал противовоспалительный эффект снижал дегрануляцию и активацию нйтрофилов посредством уменьшения уровня экспрессии молекул CD11b и CD66b на поверхности нейтрофилов, которые предварительно были активированы сыворотками пациентов с ССВО. В концентрации 3,0 мМ и выше хлорид лития индуцировал спонтанный апоптоз нейтрофилов, активированных сыворотками пациентов с ССВО после операций на сердце с ИК. The aim of this work was to study the anti-inflammatory effect of lithium chloride on human neutrophils in vitro under the action of the serum of patients with systemic inflammatory response syndrome (SIRS), which developed after on-pump cardiac surgery. Methods. The study was performed on neutrophils isolated from the blood of five healthy donors, which was activated using serum from patients with SIRS. Neutrophil activity was assessed using fluorescent antibodies to CD11b and CD66b degranulation markers. The level of apoptosis and necrosis of human neutrophils was evaluated 22 hours after isolation. Quantification was performed using annexin V and propidium iodide on a flow cytometer. Intact and activated neutrophils were treated with 0.3, 3.0 аnd 9.0 mM lithium chlorides. Results. Incubation of neutrophils with the blood serum of patients with SIRS after on-pump cardiac surgery increased the expression of CD11b by 1.5 times and the expression of CD66b by 1.4 times compared to expression on intact neutrophils. Incubation of neutrophils with blood serum of patients with SIRS and 3.0 and 9.0 mM lithium chloride solutions led to a statistically significant decrease in the level of expression of CD11b CD66b on the surface of neutrophils in comparison with control activated neutrophils. It was found that 3.0 and 9.0 mM lithium chloride solutions returned the expression levels of CD11b and CD66b on activated neutrophils to the expression level on intact neutrophils. 0.3 mM of lithium chloride, used during incubation with activated neutrophils, did not cause a significant decrease in the expression of CD11b and CD66b relative to control activated neutrophils. The expression of CD11b and CD66b on activated neutrophils during their incubation with 0.3 mM of lithium chloride was significantly higher relative to the expression of these molecules on intact neutrophils. The serum of patients with advanced SIRS decreased the ability of neutrophils to spontaneous apoptosis. 3.0 or 9.0 mM lithium chloride solutions added to the incubation medium increased the ability of neutrophils to spontaneous apoptosis. Conclusion. Lithium chloride reduced the degranulation and activation of neutrophils by reducing the expression level of CD11b and CD66b molecules on the surface of neutrophils that were previously activated by the serum of patients with SIRS. This effect determines the anti-inflammatory influence of lithium chloride. Lithium chloride at 3.0 mM and higher induced spontaneous apoptosis of neutrophils activated by the serum of patients with SIRS after on-pump cardiac surgery.