Expression Of Bcl-2 Family Members Research Articles

Inhibition of Bcl-2 family members sensitizes mesenchymal chondrosarcoma to conventional chemotherapy: report on a novel mesenchymal chondrosarcoma cell line

Mesenchymal chondrosarcomas are rare and highly aggressive sarcomas occurring in bone and soft tissue, with poor overall survival. Bcl-2 expression was previously shown to be upregulated in mesenchymal chondrosarcomas. We here report on a newly derived mesenchymal chondrosarcoma cell line, MCS170, in which we investigated treatment with the BH3 mimetic ABT-737 alone or in combination with conventional chemotherapy as a possible new therapeutic strategy. The presence of the characteristic HEY1-NCOA2 fusion was confirmed in the MCS170 cell line using FISH, RT-PCR, and sequencing. The MCS170 cell line was treated with ABT-737 alone or in combination with doxorubicin or cisplatin. Cell viability and proliferation was determined using WST-1 viability assays and the xCELLigence system. Expression of Bcl-2 family members was studied using immunohistochemistry. Apoptosis was determined using the caspase-glo 3/7 assay and western blot for PARP cleavage. The MCS170 cell line was sensitive to doxorubicin treatment with an IC50 of 0.09 μM after 72 h, but more resistant to cisplatin treatment with an IC50 of 4.5 μM after 72 h. Cells showed little sensitivity toward ABT-737 with an IC50 of 1.8 μM after 72 h. Combination treatments demonstrated ABT-737 synergism with cisplatin as well as doxorubicin as shown by induction of apoptosis and reduction in cell proliferation. Restoration of the apoptotic machinery by inhibition of Bcl-2 family members sensitizes MCS170 mesenchymal chondrosarcoma cells to conventional chemotherapy. This indicates that combining the inhibition of Bcl-2 family members with conventional chemotherapy can be a possible therapeutic strategy for patients with mesenchymal chondrosarcoma.

Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation.

Ewing sarcoma (ES) is the second most frequent childhood bone cancer driven by the EWS/FLI1 (EF) fusion protein. Genetically defined ES models are needed to understand how EF expression changes bone precursor cell differentiation, how ES arises and through which mechanisms of inhibition it can be targeted. We used mesenchymal Prx1-directed conditional EF expression in mice to study bone development and to establish a reliable sarcoma model. EF expression arrested early chondrocyte and osteoblast differentiation due to changed signaling pathways such as hedgehog, WNT or growth factor signaling. Mesenchymal stem cells (MSCs) expressing EF showed high self-renewal capacity and maintained an undifferentiated state despite high apoptosis. Blocking apoptosis through enforced BCL2 family member expression in MSCs promoted efficient and rapid sarcoma formation when transplanted to immunocompromised mice. Mechanistically, high BCL2 family member and CDK4, but low P53 and INK4A protein expression synergized in Ewing-like sarcoma development. Functionally, knockdown of Mcl1 or Cdk4 or their combined pharmacologic inhibition resulted in growth arrest and apoptosis in both established human ES cell lines and EF-transformed mouse MSCs. Combinatorial targeting of survival and cell cycle progression pathways could counteract this aggressive childhood cancer.

TRPA1 deficiency is protective in cuprizone-induced demyelination-A new target against oligodendrocyte apoptosis.

Multiple sclerosis is a chronic inflammatory, demyelinating degenerative disease of the central nervous system. Current treatments target pathological immune responses to counteract the inflammatory processes. However, these drugs do not restrain the long-term progression of clinical disability. For this reason, new therapeutic approaches and identification of novel target molecules are needed to prevent demyelination or promote repair mechanisms. Transient Receptor Potential Ankyrin 1 (TRPA1) is a nonselective cation channel with relatively high Ca2+ permeability. Its pathophysiological role in central nervous system disorders has not been elucidated yet. In the present study, we aimed to assess the distribution of TRPA1 in the mouse brain and reveal its regulatory role in the cuprizone-induced demyelination. This toxin-induced model, characterized by oligodendrocyte apoptosis and subsequent primary demyelination, allows us to investigate the nonimmune aspects of multiple sclerosis. We found that TRPA1 is expressed on astrocytes in the mouse central nervous system. Interestingly, TRPA1 deficiency significantly attenuated cuprizone-induced demyelination by reducing the apoptosis of mature oligodendrocytes. Our data suggest that TRPA1 regulates mitogen-activated protein kinase pathways, as well as transcription factor c-Jun and a proapoptotic Bcl-2 family member (Bak) expression resulting in enhanced oligodendrocyte apoptosis. In conclusion, we propose that TRPA1 receptors enhancing the intracellular Ca2+ concentration modulate astrocyte functions, and influence the pro or anti-apoptotic pathways in oligodendrocytes. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. GLIA 2016;64:2166-2180.

Selective Impairment of TH17-Differentiation and Protection against Autoimmune Arthritis after Overexpression of BCL2A1 in T Lymphocytes.

The inhibition of apoptotic cell death in T cells through the dysregulated expression of BCL2 family members has been associated with the protection against the development of different autoimmune diseases. However, multiple mechanisms were proposed to be responsible for such protective effect. The purpose of this study was to explore the effect of the T-cell overexpression of BCL2A1, an anti-apoptotic BCL2 family member without an effect on cell cycle progression, in the development of collagen-induced arthritis. Our results demonstrated an attenuated development of arthritis in these transgenic mice. The protective effect was unrelated to the suppressive activity of regulatory T cells but it was associated with a defective activation of p38 mitogen-activated protein kinase in CD4+ cells after in vitro TCR stimulation. In addition, the in vitro and in vivo TH17 differentiation were impaired in BCL2A1 transgenic mice. Taken together, we demonstrated here a previously unknown role for BCL2A1 controlling the activation of CD4+ cells and their differentiation into pathogenic proinflammatory TH17 cells and identified BCL2A1 as a potential target in the control of autoimmune/inflammatory diseases.

Abstract 430: Novel biomarkers for VEGFR inhibitors in metastatic renal cell carcinoma: BIM expression, and germline polymorphisms of BIM and PIK3R1

Abstract Molecular targeted therapy, especially VEGFR tyrosine-kinase inhibitors (TKIs) including sunitinib, pazopanib and sorafenib has been the most efficacious therapy for metastatic renal cell carcinoma (mRCC). However, approximately 35% of mRCC patients do not get the benefit from TKIs treatment, due to resistance and/or drug induced toxicities. Pro-apoptotic Bcl-2 family member (BIM) is a particularly critical mediator of targeted therapy-induced apoptosis in solid tumors. In previous, it had known that TKIs induced to BIM upregulation and then accelerated to apoptosis of tumor. However, according to recent several studies, BIM germline deletion polymorphism was identified as one of the primary resistant mechanisms of various TKIs in EGFR mutated NSCLC or CML. In addition, the alteration of phosphoinositide 3 kinase(PIK3)/protein kinase B(AKT) pathway has also been found as important mechanism in various cancer cell growth, proliferation and survival. PI3KR1 encodes the p85α subunit, regulatory subunits of Class IA PIK3s. Germline PIK3R1 variant M326I(c.978G>A) might affect the sensitivity of receptor tyrosine kinase (RTK) inhibitors and its downstream pathway inhibitors. We evaluated the possibility of BIM, PIK3R1 as a potential biomarker for VEGFR TKIs in mRCC. BIM deletion polymorphism status of gDNA from formalin fixed paraffin embedded (FFPE) tumor tissues and peripheral blood mononucleated cells (PBMC) of mRCC patients was determined by separated PCR. And we evaluated BIM protein expression by immunohistochemistry. We also evaluated PIK3R1 germline mutation by pyrosequencing in PBMC of mRCC patients.Germline BIM deletion accounted for 17.8% of total 124 mRCC patients. Unlike with previous other studies, our data suggested that BIM wild type showed tendency to poor prognosis with sunitinib or sorafenib treatments for clear cell type RCC (median OS of BIM wild and deleted type: 44.9 vs 67.3 months, p = 0.279), and high expression of BIM (median OS of BIM low(<10%), median(10-50%) and high(>50%) respectively: 76.5 vs 60.0 vs 21.3 months, p = 0.005). In addition, we found 26% (19/71) of the germline PIK3R1 variant in mRCC. Patients with PIK3R1 germline variant showed a tendency of poor prognosis to 1st line VEGFR TKI treatment than wild type (median PFS: 7.78 vs 15.87months, p = 0.342). Especially PFS of 1st line sunitinib treatment was significantly shorter in PIK3R1 variant compared to wild type (median PFS 7.78 vs 16.33 months, p = 0.004). But, there is no difference in mTOR inhibitor sensitivity both with PIK3R1 or BIM polymorphism.In conclusion, BIM expression and germline variants of BIM and PIK3R1 might be candidate biomarkers for VEGFR TKIs in mRCC. Citation Format: Jee Hung Kim, Woo Sun Kwon, Won Suk Lee, Tae Soo Kim, Kyu Hyun Park, Jae Kyung Roh, Joong Bae Ahn, Hyun Cheol Chung, Sun Young Rha. Novel biomarkers for VEGFR inhibitors in metastatic renal cell carcinoma: BIM expression, and germline polymorphisms of BIM and PIK3R1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 430.

Abstract CT069: Correlative biomarkers of clinical activity of the BCL-2 inhibitor, venetoclax (ABT-199/ GDC-0199), in acute myeloid leukemia patients

Abstract A phase 2 trial evaluated the efficacy and safety of venetoclax monotherapy in 32 patients (pts) with relapsed/refractory acute myeloid leukemia (AML) or those unfit for intensive therapy. Venetoclax demonstrated activity and a tolerable safety profile in these pts. We evaluated potential clinical biomarkers that correlated with venetoclax sensitivity and/or intrinsic/acquired resistance. Given the selectivity of venetoclax to inhibit BCL-2, protein expression of BCL-2 family members BCL-2 and BCL-XL was determined by flow cytometry in tumor cells from peripheral blood collected at baseline (22 pts) and post venetoclax therapy (21 pts). Six pts had a sensitive protein index (?35% of blast expressing BCL-2 protein and <40% blasts expressing BCL-XL) prior to therapy and the remaining 16 pts had a resistant protein index (7 pts had <35% of blasts expressing BCL-2, 9 pts had ?40% blasts expressing BCL-XL). The median time on venetoclax therapy was 30 days for pts with a resistant index vs 104 days for pts with a sensitive index (P = 0.0381, Wilcoxon). AML blasts analyzed at relapse demonstrated a resistant BCL-2/BCL-XL protein index in 20 of 21 pts analyzed: 9 pts had <35% blasts with BCL-2 expression and 11 pts had ?40% blasts with BCL-XL protein expression. All 6 pts with a sensitive protein index prior to therapy expressed a resistant protein index at relapse. Occurrence of genetic mutations known to be associated with myeloid malignancies was investigated by next generation sequencing on blood and bone marrow specimens collected at baseline (32 pts) and relapse (20 pts). Biological activity defined as any measurable reduction in bone marrow blast counts after initiation of venetoclax therapy was observed in 17 pts. Eleven of 17 (65%) pts with biological activity had blasts containing mutations in the splicing factor genes SRSF2/ZRSR2, among them 6 pts also had blasts with mutations in IDH1/IDH2. Of the remaining 6 pts with biological activity, 1 pt had an IDH2 mutation and the remaining 5 pts were wild-type for SRSF2/ZRSR2/IDH1/IDH2. Six of 15 pts without biological response to venetoclax had blasts with either FLT3-ITD or PTPN11 mutation prior to therapy. Five of the 17 pts with initial biological response to venetoclax acquired a new FLT3-ITD or PTPN11 mutation at loss of response. Previous studies have shown that both these genetic alterations affect the expression of BCL-2 family members, suggesting that FLT3-ITD or PTPN11 mutations may be responsible for primary and acquired resistance to venetoclax in AML pts by deregulating anti-apoptotic proteins. These findings suggest that BCL-2 protein expression patterns and/or presence of genetic mutations including splicing factors, IDH, PTPN11, and FLT3 may predict venetoclax monotherapy activity in pts with relapsed AML. [R.P. and N.D. contributed equally to this work.] Citation Format: Relja Popovic, Naval Daver, Vivian Ruvolo, Ken Chen, Zixing Wang, Xin Huang, Mack Mabry, Jalaja Potluri, Evelyn McKeegan, Marina Konopleva, Brenda J. Chyla. Correlative biomarkers of clinical activity of the BCL-2 inhibitor, venetoclax (ABT-199/ GDC-0199), in acute myeloid leukemia patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT069.

Abstract 3572: AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models

Abstract Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, can be indirectly modulated through transient inhibition of Cdk9 due to it having a short-lived transcript and being a labile protein. Transient inhibition of Cdk9, therefore, represents a potential therapeutic opportunity in tumors dependent on Mcl1 for survival, including various hematological malignancies. AZ5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 <5nM and decreases phosphorlyation of Ser2-RNAPII in cells with an IC50 of 96nM. In the MLL-fusion containing acute myeloid leukemia (AML) line, MV411, short-term treatment with AZ5576 led to a rapid dose-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in the cleavage and activation of caspase 3 and loss of cell viability. However, protein expression of other Bcl2 family members (e.g. Bcl2, BclxL, Bim) remained unchanged. Further, AZ5576 induced rapid caspase activation and loss of viability in a diverse set of hematological cancer cell lines, including those from acute myeloid leukemia (16/20), multiple myeloma (10/19), and diffuse large B-cell lymphoma (8/13). In vivo efficacy with intermittent dosing of AZ5576 was also demonstrated in multiple xenograft models from each hematological tumor type. Finally, AZ5576 was still able to induce loss of viability in various multiple myeloma lines cultured in the presence of cytokines or bone marrow stromal cells that represent potential protective effects of the tumor microenvironment. Together, these results highlight the therapeutic potential for selective CDK9 inhibition in the treatment of hematological malignancies. Citation Format: Justin Cidado, Minhui Shen, Michael Grondine, Scott Boiko, Haiyun Wang, Alexandra Borodovsky, Anne Marie Mazzola, Alan Wu, Deborah Lawson, Douglas Ferguson, Beirong Gao, Andy Cui, Celina D’Cruz, Lisa Drew. AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3572.

Artonin E Induces Apoptosis via Mitochondrial Dysregulation in SKOV-3 Ovarian Cancer Cells.

Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell lineT1074, with IC50 value of 32.5±0.5μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.

Inhibition of MARCH5 ubiquitin ligase abrogates MCL1-dependent resistance to BH3 mimetics via NOXA.

BH3 mimetic compounds induce tumor cell death through targeted inhibition of anti-apoptotic BCL2 proteins. Resistance to one such compound, ABT-737, is due to increased levels of anti-apoptotic MCL1. Using chemical and genetic approaches, we show that resistance to ABT-737 is abrogated by inhibition of the mitochondrial RING E3 ligase, MARCH5. Mechanistically, this is due to increased expression of pro-apoptotic BCL2 family member, NOXA, and is associated with MARCH5 regulation of MCL1 ubiquitylation and stability in a NOXA-dependent manner. MARCH5 expression contributed to an 8-gene signature that correlates with sensitivity to the preclinical BH3 mimetic, navitoclax. Furthermore, we observed a synthetic lethal interaction between MCL1 and MARCH5 in MCL1-dependent breast cancer cells. Our data uncover a novel level at which the BCL2 family is regulated; furthermore, they suggest targeting MARCH5-dependent signaling will be an effective strategy for treatment of BH3 mimetic-resistant tumors, even in the presence of high MCL1.

Inhibition of deubiquitinases primes glioblastoma cells to apoptosis in vitro and in vivo.

It remains a challenge in oncology to identify novel drug regimens to efficiently tackle glioblastoma, the most common primary brain tumor in adults. Here, we target deubiquitinases for glioblastoma therapy by utilizing the small-molecule inhibitor WP1130 which has been characterized as a deubiquitinase inhibitor that interferes with the function of Usp9X. Expression analysis data confirm that Usp9X expression is increased in glioblastoma compared to normal brain tissue indicating its potential as a therapeutic. Consistently, increasing concentrations of WP1130 decrease the cellular viability of established, patient-derived xenograft (PDX) and stem cell-like glioblastoma cells. Specific down-regulation of Usp9X reduces viability in glioblastoma cells mimicking the effects of WP1130. Mechanistically, WP1130 elicits apoptosis and increases activation of caspases. Moreover, WP1130 and siRNAs targeting Usp9X reduce the expression of anti-apoptotic Bcl-2 family members and Inhibitor of Apoptosis Proteins, XIAP and Survivin. Pharmacological and genetic interference with Usp9X efficiently sensitized glioblastoma cells to intrinsic and extrinsic apoptotic stimuli. In addition, single treatment with WP1130 elicited anti-glioma activity in an orthotopic proneural murine model of glioblastoma. Finally, the combination treatment of WP1130 and ABT263 inhibited tumor growth more efficiently than each reagent by its own in vivo without detectable side effects or organ toxicity. Taken together, these results suggest that targeting deubiquitinases for glioma therapy is feasible and effective.

Plumbagin shows anti-cancer activity in human breast cancer cells by the upregulation of p53 and p21 and suppression of G1 cell cycle regulators.

Plumbagin, a naphthoquinone constituent of Plumbago zeylanica L. (Plumbaginaceae), is known to exhibit proapoptotic, antiangiogenic and antimetastatic effects in cancer cells. However, the effect of Plumbagin on breast cancer cells and the underlying molecular mechanism has not yet been elucidated. MCF-7 (a human breast cancer cell line) was exposed different concentrations of Plumbagin (PG), and the anti-proliferative activity was evaluated by the MTT assay. The mechanism of action for the growth inhibitory activity of Plumbagin on MCF-7 cancer cells was evaluated using flow cytometry for cell cycle distribution, and western blot for assessment of expression of potential target proteins. Plumbagin exhibited a significant anti-proliferative activity against human breast cancer cells. Flow cytometric analysis revealed that Plumbagin caused cell cycle arrest at G1 phase. The cell cycle arrest was well correlated with the inhibition of cyclin D1, cyclin E, and upregulation of tumor suppressor protein p53. It further inhibited the expression of anti-apoptotic Bcl-2 family members such as Bcl-xL and Bcl-2, and activated pro-apoptotic proteins like Bax and Bak. These findings suggest that the anti-proliferative effect of Plumbagin is due to upregulation of p53 and p21 and suppression of G1 cell cycle regulators.

Bcl-2 expression is a poor predictor for hepatocellular carcinoma prognosis of andropause-age patients.

Objective: The expression of B-cell lymphoma 2 (Bcl-2) seems to be influenced by the endocrine environment. Numerous reports demonstrate the diverse expression of Bcl-2 family members under sex steroid regulation. With the exception of estrogen-related tumors, androgen-related tumors have shown their characteristics in Bcl-2 expression. In this study, the status of Bcl-2 expression in male hepatocellular carcinoma (HCC) patients was examined to verify the high incidence of HCC in males.Methods: Tumor tissue microarray was used to examine Bcl-2 expression levels in 374 HCC cases including 306 males and 68 females. Kaplan-Meier method, log-rank test, and Cox proportional hazards model were applied to investigate the predictive value of Bcl-2 in HCC patients.Results: Immunohistochemistry analysis showed that male patients with higher Bcl-2 levels had significantly longer median survival time and recurrence time than those with lower levels. However, no significant differences in outcomes were found between different Bcl-2 levels in female patients. When the male patients were stratified into several age points, the level of Bcl-2 expression showed poorer predictive efficiency in the 45–49 and 55–60 age groups in andropause-age patients compared with other age groups. Bcl-2 was an independent prognostic factor for both overall survival (P < 0.0001) and recurrence time (P = 0.0001) in male patients. After excluding male patients in the 45–60 age group, the predictive efficiency was enhanced (n = 147, OS, P = 0.0002, TTR, P < 0.0001). Conclusions: Bcl-2 expression is an independent predictor of survival and recurrence in male HCC. Bcl-2 levels may also be regulated by androgens or androgen receptors in male HCC patients. Bcl-2 levels change and exhibit poor predictive efficiency when androgen levels vary dramatically (andropause age).

BCL2-Inhibitors Target a Major Group of Newly-Diagnosed and Relapsed/Refractory Acute Myeloid Leukemia Ex Vivo

BACKGROUND:BCL-2 family members play a critical role in the regulation of apoptosis. BCL-2 and BCL-XL promote cell survival by preventing mitochondrial apoptotic pore formation. BH3 mimetic drugs such as venetoclax (ABT-199) promote apoptosis by inhibiting BCL-2 while navitoclax (ABT-263) inhibits both BCL-2 and BCL-XL. In AML, the expression of anti-apoptotic proteins is highly variable. In a recent study venetoclax showed single-agent activity in 6/12 AML cell lines and 20/25 patient samples. (Pan et al. Cancer Disc 2014). The samples with complex cytogenetics were largely resistant. Sensitivity correlated with increased BCL-2 protein levels and negatively correlated with BCL-XL and MCL-1 protein levels. We aimed to expand these data to both newly diagnosed and relapsed AML patients and to isolate biomarkers for patient selection.METHODS:We assessed the ex vivo sensitivity of fresh leukemic cells from 16 diagnosed and 36relapsed/refractory AML patient samples to venetoclax (25 samples) and navitoclax (52 samples). Exome sequencing was performed on 32 samples and gene expression of BCL2 family members (BCL-2, BCL-XL, MCL-1, BIK, BAX, BAK1, BID, BCL2L12, BIM, BCL2A1, PUMA and BAD) was determined on 31 samples by qRT-PCR. Samples from primary cells of healthy individuals (n=10), and CMML (n=7) or CLL (n=2) patients were used as controls. Drug sensitivity was determined over a 10,000-fold concentration range (1-10 000 nM). A leukemia-specific drug sensitivity score (sDSS) derived from area under the dose response curve calculations was used as the efficacy variable by comparing leukemia results with those from normal bone marrow cells (Bhagwan et al., Sci Rep 2014, Pemovska et al., Cancer Disc 2014).RESULTS:Compared to healthy controls, CMML samples were largely non-sensitive, whereas CLL samples were highly sensitive to BCL-2 inhibitors ex vivo. The AML samples exhibited heterogeneous responses. 15/25 (60%) of AML samples were sensitive to venetoclax and 35/52 (67%) to navitoclax.Both diagnostic (12 of 16 samples, 75%) and relapsed/refractory samples (24 of 36 samples, 64%) were sensitive to navitoclax. Similarly, 6/7 (86%) of diagnostic samples and 9/18 (50%) of relapsed/refractory samples were sensitive to venetoclax. We observed responses to venetoclax and navitoclax in each patient to be similar, although navitoclax showed efficacy at lower concentrations: in 25 samples tested with both agents, mean sDSS values were lower in navitoclax-treated samples (paired t-test, p=0.02). All except one patient sample exhibited a difference in resistance between the two drugs showing sensitivity to navitoclax but not to venetoclax.We observed responses across all mutational profiles, including samples with mutations to FLT3 -ITD, NPM1, TP53, NRAS and IDH1 and IDH2, as well as in samples with complex karyotypes. Intriguingly, three of four samples with mutated TP53 exhibited sensitivity to BCL2 inhibition. No single mutation predicted sensitivity or resistance. At the RNA level, no statistical correlation between BCL2 or BCL-XL expression for BCL2 inhibitor response was observed. Instead we observed high levels of beta-2-microglobulin (B2M) mRNA expression in BCL2 inhibitor-resistant samples with a strong negative correlation to navitoclax sensitivity (r=-0.60, P=0.0008).DISCUSSION:We did not observe BCL2 and BCL-XL mRNA expression to be optimal predictors for BCL2 inhibitor response. On the other hand, we observed high expression of B2M mRNA expression in resistant samples suggesting that it could serve as a biomarker for sensitivity to BCL2 inhibitors. The high B2M expression has been previously linked to poor prognosis in solid tumors and in AML (Albitar et al., Leukemia 2007). In cell line models B2M leads to phosphorylation and inactivation of proapototic protein BAD (Nokura et al., J Urol 2007). This may affect the balance between pro- and antiapoptotic proteins and thus offer a escape route from BCL2 inhibition.To conclude, we observed BCL2 inhibition to be effective ex vivo in over half of all AML samples, tested both in primary and relapsed/refractory state as well as across different subgroups defined by AML driver mutations. Of the potential biomarkers that were assessed, B2M was the best mRNA-level indicator for anti-BCL-2 drug efficacy. DisclosuresOff Label Use: BCL2 inhibitors are not approved for the treatment of AML. Heckman:Celgene: Honoraria, Research Funding; Pfizer: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Induction of Resistance to Proteasome Inhibition Preferentially Switches Survival Dependence from Bcl-2 to XIAP in Preclinical Models of Waldenstrom Macroglobulinemia: Pre-Clinical Rationale for Early Clinical Sequencing of ABT199

Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance.Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem.Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B).Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca2+ concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.

CD73 is associated with poor prognosis in high-grade serous ovarian cancer.

The cell surface nucleotidase CD73 is an immunosuppressive enzyme involved in tumor progression and metastasis. Although preclinical studies suggest that CD73 can be targeted for cancer treatment, the clinical impact of CD73 in ovarian cancer remains unclear. In this study, we investigated the prognostic value of CD73 in high-grade serous (HGS) ovarian cancer using gene and protein expression analyses. Our results demonstrate that high levels of CD73 are significantly associated with shorter disease-free survival and overall survival in patients with HGS ovarian cancer. Furthermore, high levels of CD73 expression in ovarian tumor cells abolished the good prognosis associated with intraepithelial CD8(+) cells. Notably, CD73 gene expression was highest in the C1/stromal molecular subtype of HGS ovarian cancer and positively correlated with an epithelial-to-mesenchymal transition gene signature. Moreover, in vitro studies revealed that CD73 and extracellular adenosine enhance ovarian tumor cell growth as well as expression of antiapoptotic BCL-2 family members. Finally, in vivo coinjection of ID8 mouse ovarian tumor cells with mouse embryonic fibroblasts showed that CD73 expression in fibroblasts promotes tumor immune escape and thereby tumor growth. In conclusion, our study highlights a role for CD73 as a prognostic marker of patient survival and also as a candidate therapeutic target in HGS ovarian cancers.

Synergistic effects of p53 activation via MDM2 inhibition in combination with inhibition of Bcl-2 or Bcr-Abl in CD34+ proliferating and quiescent chronic myeloid leukemia blast crisis cells.

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53's ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

Targeting Galectin As A Strategy To Improve BH3 Mimetic Efficacy For The Therapy Of Acute Myeloid Leukemia

Background: Galectins are a family of glycan binding proteins that regulate cell survival by mechanisms including apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus the stability of proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, galectin 3 (LGALS3) expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013). Current strategies to eradicate leukemia cells involve use of agents targeting BCL2 family members such a BH3 mimetics ABT-7373 and ABT-199. MCL-1 has been implicated as a resistance factor against BH3 mimetic killing and unfortunately an effective anti-MCL-1 drug is currently not available for use in the clinic. Alternatively, a strategy to target MCL-1 indirectly by suppressing Galectin 3 could improve BH3 mimetic killing of leukemia cells. In the current study we examine the effect of Galectin 3 suppression by shRNA or with a broad Galectin inhibitor on BH3 mimetic induced apoptosis in AML cell lines OCI-AML3 and THP1. Study Design: GCS-100 (kind gift from George Tidmarsh; La Jolla Pharmaceutical, San Diego, CA), is now in a Phase II clinical trial for chronic kidney disease. The drug was used in combination with BH3 mimetic ABT-737 (which targets BCL2 and BCL-XL at low dose). Galectin 3 was suppressed in both AML cell lines using lentiviral shRNA. Effect of GCS-100/ABT-737 combination was tested in an in vitro model of the tumor microenvironment using mesenchymal stem cells (MSC). Cell growth and induction of apoptosis was measured by flow cytometry. Analysis of protein expression and signaling was assessed by immunoblot analysis. Level of gene expression of various BCL2 family members was measured by qRT-PCR. Results: As a single agent GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60. The drug potently suppresses phosphorylation of AKT and ERK and reduces protein expression of BCL2 and MCL-1. We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by w 30% and induced apoptosis in 80% and > 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective preventing leukemia cell contact with MSC. Conclusions: Our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy.

Abstract 2923: A novel cell-penetrating peptide targeting ATF5 (CP-d/n-ATF5) exerts anti-cancer activity in vitro and in vivo against a broad spectrum of human cancers

Abstract Background The purpose of this study was to validate the anti-neoplastic activity of a novel ATF5-targeted therapeutic approach against various recalcitrant human malignancies. Activating transcription factor 5 (ATF5) is a member of the cAMP response element binding (CREB)/ATF subfamily of basic leucine zipper transcription factors and has been shown to be overexpressed in many human cancers. CP-d/n-ATF5 is a novel peptide specifically designed to contain a cell-penetrating domain and a domain inhibiting the function of ATF5 in a unique and novel targeted therapeutic approach. This therapeutic strategy was tested among a large panel of different therapy refractory human cancers including glioblastoma, melanoma, breast, prostate-, lung- and pancreatic cancer. Methods Cellular viability was assessed by MTT-assays. The induction of apoptosis was examined by staining for annexin V/propidium iodide or JC-1 (loss of mitochondrial membrane potential) prior to flow cytometric analysis. Western blotting was performed for further molecular analysis detecting caspase cleavage and expression of Bcl-2 family members. Subcutaneous xenograft models were used to examine the anti-neoplastic effects of CP-d/n-ATF5 in vivo. Results Our data show that CP-d/n-ATF5 yields a significant increase in apoptosis across a panel of 10 different cancer cell lines when compared to treatment with a mock-mutated control peptide or penetratin. On the molecular level, treatment with CP-d/n-ATF5 resulted in a marked down-regulation of the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-2. This effect was amplified in a synergistic manner by adding either ABT-263 or TRAIL to a combined targeted therapeutic approach. In vivo, treatment with CP-d/n-ATF5 resulted in a significant reduction of tumor growth in a heterotopic glioblastoma- and an orthotopic melanoma model. Conclusions The novel ATF5-targeting compound CP-d/n-ATF5 provides a broad and pronounced anti-neoplastic activity against human cancers in vitro and in vivo. On the molecular level, this effect is at least in part due to a shift of the cellular homeostasis towards a pro-apoptotic cellular phenotype by inhibiting anti-apoptotic Bcl-2 family proteins. Overall, CP-d/n-ATF5 may represent a promising novel anti-cancer agent. Citation Format: Georg Karpel-Massler, Chang Shu, Lily Chau, James M. Angelastro, Lloyd A. Greene, Markus D. Siegelin. A novel cell-penetrating peptide targeting ATF5 (CP-d/n-ATF5) exerts anti-cancer activity in vitro and in vivo against a broad spectrum of human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2015-2923

Tamoxifen-Induced Cell Death of Malignant Glioma Cells Is Brought About by Oxidative-Stress-Mediated Alterations in the Expression of BCL2 Family Members and Is Enhanced on miR-21 Inhibition.

High-grade gliomas are refractory to the current mode of treatment primarily due to their inherent resistance to cell death. Tamoxifen has been reported to inhibit growth and induce cell death of glioma cells in vitro, in an estrogen-receptor-independent manner. Delineating the molecular mechanism underlying tamoxifen-induced cell death of human glioma cells would help in identifying pathways/genes that could be targeted to induce tumor-cell-specific cell death. In the present study, tamoxifen was found to bring about autophagic cell death of human glioma cells that was accompanied by oxidative stress induction, JNK activation, downregulation of anti-autophagic BCL2 family members, viz. BCL2 and BCL-XL, and increased expression of the pro-autophagic members BCL-Xs and BAK. Oxidative stress induction appears to be primarily responsible for the tamoxifen-induced cell death since the cell death, JNK activation, and the alterations in the expression levels of BCL2 family members were abrogated on pretreatment with antioxidant vitamin E. MiR-21, an oncogenic miRNA, is known to be highly upregulated in malignant glioma. Inhibition of miR-21 activity was found to enhance tamoxifen-induced cell death of U87 MG malignant glioma cells. Tamoxifen treatment coupled with miR-21 inhibition could therefore be an effective strategy for the treatment of malignant gliomas.