Expression Of Bcl-2 Family Members Research Articles

Abstract 63: Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members

Abstract Colorectal cancer (CRC) is the third most common cancer in the United States. Most CRCs in patients receiving therapeutic agents are resistant to apoptotic stimuli. Two protein families, caspase enzymes and B-cell lymphoma-2 (Bcl-2) family proteins are involved in apoptosis. Caspase enzymes promote apoptosis, functioning in a cascade, of which caspase 3 is the pivotal protein. Members of the Bcl-2 family, Bcl-2, Bcl-xL, and Bax, are functionally opposed: Bcl-2 and Bcl-xL inhibit apoptosis, whereas Bax counteracts this effect. Only Bcl-2 family members that have the BH3-domain trigger apoptosis by binding to the pro-survival proteins, thereby neutralizing their functional activity. The approach of “BH-3 mimetics” was utilized for discovery and development of navitoclax (ABT-263), a small-molecule that mimics BH3-only proteins and thus induces apoptosis. Despite advances made in targeted therapy, the mechanism of action of ABT-263 is less studied, particularly for CRCs, which have over-expression of anti-apoptotic proteins, supporting avoidance of cell death, a hallmark of cancer. To identify the cellular mechanism of ABT-263 on apoptosis, we quantified the expression of anti-apoptotic Bcl-2-family members in CRC tumor samples relative to their corresponding controls and found several-fold higher expression of Bcl2, Bcl-xL, and Mcl-1 in the CRC tumor samples. Since ABT-263 is a classical drug for Bcl-2 inhibition, we studied its effect (2.5 µM for 24 hr) on RKO CRC cells, which have high endogenous expression of Bcl-2, by measuring gene expression profiles of Bcl-2 family members by qPCR, along with other proteins involved in apoptosis. ABT-263 treatment resulted in down-regulation of anti-apoptotic modulators, Bcl-2, Bcl-xL, and Bcl-w, by 27%, 29%, and 13%, respectively; these effects were concomitant with two-fold higher expression of caspase 3. Flow cytometry results with Annexin V/PI staining showed that ABT-263 induced apoptosis in RKO cells with 1.59-, 2.59-, and 2.76-fold elevations of early apoptotic, late apoptotic, and necrotic cells. Further, ABT-263 (2.5 µM) lowered cell proliferation by 15% after 24 hr. Similar lower gene expressions of Bcl-2 family members were evident for LS174T CRC cells, which also exhibit high endogenous Bcl-2 expression, when treated with ABT-263. These results highlight a previously unexplored effect of ABT-263-induced apoptosis in CRC cells, by upregulating caspase 3, through inhibition of Bcl-2 family members. These studies were supported by the Richard Elkus, MD, Eminent Scholars Program in Gastro-Intestinal Cancer Research at University of Alabama at Birmingham. Citation Format: Prachi Bajpai, Amr Elkholy, Sumit Agarwal, Hyung Gyoon Kim, Michael Behring, Upender Manne. Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 63.

Chelerythrine hydrochloride inhibits proliferation and induces mitochondrial apoptosis in cervical cancer cells via PI3K/BAD signaling pathway

Cervical cancer is the fourth most common female cancer worldwide, and drug targeted therapy plays a crucial role in delaying the progression of cervical carcinoma. Chelerythrine hydrochloride (CHE) is a natural alkaloid, which is a focal point in anti-tumor research. In this study, we investigated the effect of CHE on HeLa cells by using MTT assay, RTCA, and colony formation assay. In addition, the flow cytometric analysis, immunofluorescence staining assay and western blot analysis were performed to study the mechanism of CHE. The results showed that CHE exhibited a significant inhibitory effect in HeLa cells, and it could suppress the expression of PI3K subunits in a dose-dependent manner. Moreover, we found that the treatment of CHE further restrained the downstream AKT/mTOR and PKCα signaling. In addition, CHE induced mitochondrial apoptosis of HeLa cells by regulating the BCL-2 family member's expression. Immunofluorescence staining assay indicated that AIF and Cytochrome C were translocated from mitochondria to cytoplasm or nucleus, and notable changes in mitochondrial morphology of HeLa cells were also observed. Finally, the aberrant expression of CHE led to the mitochondrial apoptosis by upregulating the expression of APAF1, Cleaved-Caspase9, Cleaved-Caspase3, and Cleaved-PARP. In summary, CHE suppresses the proliferation of HeLa cells by trigging the mitochondrial apoptosis through the PI3K/BAD signaling pathway.

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.

Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor. BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system. Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy. This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

Evaluation of combined BCL-2/MCL-1 inhibition as a therapeutic approach for synovial sarcoma.

e23561 Background: While Synovial Sarcoma (SS) only accounts for about 10% of soft tissue sarcomas (STS), or ~10,000 cases/year, ~4,000 of these will be fatal. Little benefit has been seen with newer combination chemotherapies and immune checkpoint inhibitors. SS is a transcription factor-driven cancer characterized by an t(X;18) chromosomal translocation resulting in the SS18-SSX fusion gene. SS is also known to overexpress the anti-apoptotic protein BCL-2, which has been used as a diagnostic marker for SS. Venetoclax, the FDA approved BH3 mimetic which specifically inhibits BCL-2, has been used to treat BCL-2-driven hematological cancers. Yet, preclinical studies of venetoclax, currently in clinical use for BCL-2 related hematologic malignancies, have failed in SS, raising the possibility of other BCL-2 family members playing a role in resistance. Methods: Publicly available gene expression databases of SS were queried for expression of BCL-2 family member expression, including the endogenous MCL-1 inhibitor, NOXA. Six SS cell lines, including ASKA, HS-SY-II, Yamato, SW982, SYO.1, and an ex vivo line from a SS patient-derived xenograft (PDX) were tested in vitro with combinations of venetoclax and the MCL-1 inhibitor S63845, using cell viability assays. In vivo testing employed both a patient-derived xenograft (PDX) model of SS and a cell line derived (SYO.1) xenograft model. Results: Queries of published gene expression data for SS demonstrated that SS expresses low levels of the endogenous MCL-1 inhibitor, NOXA, nominating a mechanism for intrinsic venetoclax resistance in these tumors, and suggesting rational combination of venetoclax and the MCL-1 inhibitor, S63845. In vitro, SS all cell lines possessing the SS18-SSX fusion gene demonstrated synergistic sensitivity to combined S63845 and venetoclax, despite generally insensitivity to either drug as monotherapy. Importantly, in an SS PDX, we demonstrated S63845 and venetoclax synergize to induce tumor regression. In the SYO.1 xenograft model, combination therapy significantly reduced tumor growth compared to no treatment or single agent groups. Conclusions: These findings provide the preclinical rationale for translation of the rational combination of BCL-2 and MCL-1 inhibitors into clinical trials for SS.

Perinatal taurine exerts a hypotensive effect in male spontaneously hypertensive rats and down‐regulates endothelial oxide nitric synthase in the aortic arch

Essential hypertension is considered to be a result of the interaction between genetic and environmental factors, including perinatal factors. Different advantageous perinatal factors proved to have beneficial long-lasting effects against an abnormal genetic background. Taurine is a ubiquitous sulphur-containing amino acid present in foods such as seafood. The antihypertensive effects of taurine have been reported in experimental studies and in human hypertension. We aimed to investigate the effects of perinatal treatment with taurine in spontaneously hypertensive rats (SHR), a known model of genetic hypertension. Female SHR were administered with taurine (3g/L) during gestation and lactation (SHR-TAU). Untreated SHR and Wistar-Kyoto rats (WKY) were used as controls. Long-lasting effects in offspring were investigated. Addition of taurine to the mother's drinking water reduced blood pressure in adult offspring. No differences were observed in cardiac hypertrophy. Findings on morphometric evaluations suggest that perinatal treatment with taurine would be partially effective in improving structural alterations of the aorta. Modifications in gene expression of Bcl-2 family members and upregulation of endothelial nitric oxide synthase in the aorta of 22-week-old male offspring were found. No differences were observed on relative telomere length in different cardiovascular tissues between SHR and SHR-TAU. Altogether results suggest that taurine programming, albeit sex specific, is associated with gene expression changes which ultimately may lead to improvement of aortic remodelling and enhanced endothelial function because of augmented nitric oxide (NO) production.

Role of Bcl-2 on drug resistance in breast cancer polyploidy-induced spindle poisons.

Spindle poisons are chemotherapeutic drugs used in the treatment of malignant tumors; however, numerous patients develop resistance following chemotherapy. The present study aimed to induce polyploidy in breast cancer cells using the spindle poison nocodazole to investigate the mechanism of polyploid-induced tumor resistance. It was revealed that the spindle poison nocodazole induced apoptosis in HCC1806 cells but also induced polyploidy in MDA-MB-231 cells. The drug sensitivities of the polyploid MDA-MB-231 cells to paclitaxel, docetaxel, epirubicin, 5-fluorouracil and oxaliplatin were lower than those of the original tumor cells; however, the polyploid MDA-MB-231 cells were more sensitive to etoposide than the original tumor cells. The expression of F-box and WD repeat domain containing 7 (FBW7) was decreased, while the expression of MCL1 apoptosis regulator BCL2 family member (MCL-1) and Bcl-2 was increased, and caspase-3/9 and Bax were not expressed in MDA-MB-231 cells. The resistance to docetaxel and etoposide was reversed, but the sensitivity of paclitaxel was not changed following Bcl-2 silencing. The formation of polyploidy in tumors may be one of the molecular mechanisms underlying tumor resistance to spindle poisons. Expression of the Bcl-2 family members, for example FBW7 and MCL-1, plays a key role in apoptosis and the cell escape process that forms polyploid cells. However, Bcl-2 silencing has different reversal effects on different anti-tumor drugs, which requires further investigation.

The Combination of Dexamethasone and Ruxolitinib Synergistically Attenuates Disease Manifestations in a Preclinical Model of Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a severe immunologic syndrome characterized by a failure of CD8 T-cells to appropriately terminate immune responses, leading to excessive activation of immune cells that mediate life-threatening organ damage. Many patients with HLH are refractory to front-line therapy consisting primarily of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide, resulting in five-year survival rates of only 62%. Therefore, novel strategies are needed to enhance the efficacy of frontline therapy in order to improve clinical outcomes. Hypercytokinemia is a hallmark feature of HLH due to the persistent activation of immune cells. CD8 T-cells both secrete and respond to these cytokines, making them important cellular targets of HLH therapy. Many HLH-associated cytokines activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine models of HLH and in clinical case reports of refractory HLH. Importantly, JAK/STAT signaling has been shown in other disease contexts to induce DEX resistance. We therefore hypothesized that cytokine-mediated JAK/STAT signaling might contribute to DEX resistance in HLH and that this could be overcome by combination treatment with RUX. To test this hypothesis, we infected Prf1-/- mice with lymphocytic choriomeningitis virus (LCMV) to generate an in vivo model of primary HLH. Beginning on day four post-infection, mice were treated with vehicle control, DEX, RUX, or the combination of DEX and RUX and examined for signs of HLH. The Bliss independence model of synergy was applied to quantify the combinatorial effects of these drugs on disease parameters. Combined treatment with DEX and RUX synergistically lessened signs of systemic inflammation, including splenomegaly, numbers of inflammatory cells, including neutrophils and CD8 T-cells, and circulating levels of inflammatory cytokines. The mechanistic basis for these findings was then interrogated ex vivo. First, activated murine and human CD8 T-cells were exposed to DEX or etoposide in the presence of HLH-associated cytokines and were evaluated for cell viability. This revealed that the JAK-dependent cytokines IL-2 and IL-12 confer resistance specifically to DEX, but not to etoposide. IL-2 and IL-12 receptor signaling converge to activate STAT5. Consistent with this, exposure to RUX attenuated STAT5 activation in response to IL-2 or IL-12 stimulation. Furthermore, DEX and RUX synergized to induce cell death in the presence of IL-2 or IL-12. Mechanistic studies revealed that cytokine exposure did not inhibit nuclear translocation of ligand-activated glucocorticoid receptor (GR) or activation of GR transcriptional targets, suggesting that IL-2 and IL-12 act downstream of GR activity to confer DEX resistance. To interrogate this further, we quantified expression of BCL-2 family members in CD8 T-cells exposed to IL-2 or IL-12 and found significant upregulation of BCL-2 and BCL-XL. Using BH3 profiling, we functionally interrogated the intrinsic apoptotic pathway and found that cytokine exposure significantly suppressed the apoptotic potential of CD8 T-cells, such that DEX alone was no longer sufficient to induce apoptotic priming. However, concomitant exposure to RUX effectively restored apoptotic priming in response to DEX by inhibiting cytokine-induced upregulation of BCL-2 and BCL-XL. Finally, we performed BH3 profiling on cells from Prf1-/- mice following in vivo treatment with DEX +/- RUX. Similar to the ex vivo findings, LCMV infection and the subsequent onset of HLH significantly reduced the apoptotic potential of CD8 T-cells relative to cells from naïve mice, consistent with elevated levels of circulating cytokines in vivo. Combined treatment with DEX and RUX was more effective than either agent alone at inducing apoptotic priming, suggesting that both ex vivo and in vivo, cytokine exposure inhibits DEX-induced cell death by altering the cellular apoptotic potential. Taken together, this study reveals a mechanism of cytokine-mediated DEX resistance in HLH and provides rationale for combining DEX and RUX as a means to augment DEX sensitivity and improve clinical outcomes for patients with HLH. Figure Disclosures Nichols: Incyte: Research Funding.

The Effect of Apoptosis Inhibitor Blockade Agents on the Third Generation CD19 CAR T Cells

Background: Chimeric antigen receptor (CAR) engineered T cell therapy is currently revolutionizing the field of cancer immunotherapy. However, further enhancement of the function of CAR T cells either through modification of CAR structure or through combination with other therapies is intensively investigated. Resistance to apoptosis is one of the hallmarks of cancer biology. Overexpression of anti-apoptotic proteins of the Bcl-2 family proteins, such as Bcl-2, Mcl-1 and Bcl-xl is considered primarily responsible for the increased apoptosis resistance and prolonged survival of hematological tumor cells. Apoptosis inhibitor blockade agents (AIBAs) such as Bcl-2, Mcl-1 and COX inhibitors could sensitize malignant cells to apoptosis. Therefore, combining CAR T cell therapy with AIBAs might be a promising approach to increase the therapeutic response. However, the third generation of CAR T cells including a 4-1BB co-stimulatory domain which triggers a signaling cascade with the upregulation of anti-apoptotic molecules might be hampered by AIBAs. Therefore, in this study we analyzed the influence of AIBAs on third generation CD19 CAR T cells. Materials and methods: CD19 CAR T cells were manufactured using a 3rd generation CAR vector containing both CD28 and 4-1BB co-stimulatory domains. The expression of anti-apoptotic Bcl-2 family members in leukemia/lymphoma cell lines and in CD19 CAR T cells have been assessed by quantitative real time polymerase chain reaction (qRT-PCR), western blot (WB) and flow cytometery. The optimal concentrations of inhibitors have been determined by CellTiter Glo™ assay. The effect of inhibitors on CAR T cells and tumor cells has been evaluated by chromium-51 (51Cr) release assay, Calcein AM™ assay and FACS-based apoptosis assay. Intracellular cytokine staining and surface marker staining were performed to determine the function of CAR T cells. Results: qRT-PCR and WB showed that leukemia/lymphoma cell lines 380 and U698M had the highest Bcl-2 (qRT-PCR: 1.73 ± 0.04, p = 0.001; WB: 1.82 ± 0.54, p = 0.016) and Mcl-1 (qRT-PCR: 12.39 ± 1.37, p = 0.000; WB: 1.92 ± 1.08, p = 0.142) expression levels, respectively,. These findings were confirmed by anti-apoptotic BCL-2 family protein intracellular staining. Elevated protein expression of Mcl-1, Bcl-2 and Bcl-xl was observed in CAR T cells upon 380 cell stimulation. The manufactured CD19 CAR T cells exhibited specific killing on CD19+ tumor cells. A slightly enhanced killing efficiency of CAR T cells was observed when 380 cells were co-cultured with CAR T cells in the presence of ABT199 (0 µM vs. 1 µM: 54.21 ± 3.23 vs. 69.06 ± 2.17, p = 0.0041). This result could be explained by a higher sensitivity of 380 cells to ABT199 than CART cells and by an increased activation of CAR T cells. Even though the killing efficiency of CAR T cells was not improved by S63845, the CD107a+TNF-α+IFN-γ+ CAR T cells were increased as well as the proliferation and persistence of CAR T cells were strengthened. Of note, pretreatment of tumor cells with inhibitors could significantly enhance the killing efficiency of CAR T cells via upregulation of CD19 antigen on tumor cells (ABT199-pretreated 380 cells: 0 µM vs.1 µM, 42.59 ± 5.82 vs. 54.58 ± 4.87, p = 0.0493; S63845-pretreated-U698M cells: 0 µM vs. 0.3 µM, 31.88 ± 4.77 vs. 66.35 ± 8.09, p = 0.000). Conclusion: The quantity of CAR T cells can be negatively affected by Apoptosis inhibitor blockade agents. However, the quality of CAR T cells could be improved. The cytotoxic effect of CAR T cells can be further enhanced by pretreatment of tumor cells with inhibitors. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support.

CD27 Promotes CD4+ Effector T Cell Survival in Response to Tissue Self-Antigen.

Signaling through CD27 plays a role in T cell activation and memory. However, it is currently unknown how this costimulatory receptor influences CD4+ effector T (Teff) cells in inflamed tissues. In the current study, we used a murine model of inducible self-antigen expression in the epidermis to elucidate the functional role of CD27 on autoreactive Teff cells. Expression of CD27 on Ag-specific Teff cells resulted in enhanced skin inflammation when compared with CD27-deficient Teff cells. CD27 signaling promoted the accumulation of IFN-γ and IL-2-producing T cells in skin draining lymph nodes in a cell-intrinsic fashion. Surprisingly, this costimulatory pathway had minimal effect on early T cell activation and proliferation. Instead, signaling through CD27 resulted in the progressive survival of Teff cells during the autoimmune response. Using BH3 profiling to assess mitochondrial cell priming, we found that CD27-deficient cells were equally as sensitive as CD27-sufficient cells to mitochondrial outer membrane polarization upon exposure to either BH3 activator or sensitizer peptides. In contrast, CD27-deficient Teff cells expressed higher levels of active caspase 8. Taken together, these results suggest that CD27 does not promote Teff cell survival by increasing expression of antiapoptotic BCL2 family members but instead acts by preferentially suppressing the cell-extrinsic apoptosis pathway, highlighting a previously unidentified role for CD27 in augmenting autoreactive Teff cell responses.

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

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

LGALS3 is connected to CD74 in a previously unknown protein network that is associated with poor survival in patients with AML

BackgroundGalectin 3 (LGALS3) gene expression is associated with poor survival in acute myeloid leukemia (AML) but the prognostic impact of LGALS3 protein expression in AML is unknown. LGALS3 supports diverse survival pathways including RAS mediated cascades, protein expression and stability of anti-apoptotic BCL2 family members, and activation of proliferative pathways including those mediated by beta Catenin. CD74 is a positive regulator of CD44 and CXCR4 signaling and this molecule may be critical for AML stem cell function. At present, the role of LGALS3 and CD74 in AML is unclear. In this study, we examine protein expression of LGALS3 and CD74 by reverse phase protein analysis (RPPA) and identify new protein networks associated with these molecules. In addition, we determine prognostic potential of LGALS3, CD74, and their protein networks for clinical correlates in AML patients. MethodsRPPA was used to determine relative expression of LGALS3, CD74, and 229 other proteins in 231 fresh AML patient samples and 205 samples were from patients who were treated and evaluable for outcome. Pearson correlation analysis was performed to identify proteins associated with LGALS3 and CD74. Progeny clustering was performed to generate protein networks. String analysis was performed to determine protein:protein interactions in networks and to perform gene ontology analysis. Kaplan-Meir method was used to generate survival curves. FindingsLGALS3 is highest in monocytic AML patients and those with elevated LGALS3 had significantly shorter remission duration compared to patients with lower LGALS3 levels (median 21.9 vs 51.3 weeks, p = 0.016). Pearson correlation of LGALS3 with 230 other proteins identifies a distinct set of 37 proteins positively correlated with LGALS3 expression levels with a high representation of proteins involved in AKT and ERK signaling pathways. Thirty-one proteins were negatively correlated with LGALS3 including an AKT phosphatase. Pearson correlation of proteins associated with CD74 identified 12 proteins negatively correlated with CD74 and 16 proteins that are positively correlated with CD74. CD74 network revealed strong association with CD44 signaling and a high representation of apoptosis regulators. Progeny clustering was used to build protein networks based on LGALS3 and CD74 associated proteins. A strong relationship of the LGALS3 network with the CD74 network was identified. For AML patients with both the LGALS3 and CD74 protein cluster active, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. InterpretationThe findings from this study identify for the first time protein networks associated with LGALS3 and CD74 in AML. Each network features unique pathway characteristics. The data also suggest that the LGALS3 network and the CD74 network each support AML cell survival and the two networks may cooperate in a novel high risk AML population. FundLeukemia Lymphoma Society provided funds to SMK for RPPA study of AML patient population. Leukemia Texas provided funds to PPR and SMK to study CD74 and LGALS3 expression in AML patients using RPPA. No payment was involved in the production of this manuscript.

The interaction between Vav1 and EBNA1 promotes survival of Burkitt's lymphoma cells by down-regulating the expression of Bim

Vav1 is a guanine nucleotide exchange factor (GEF) predominantly expressed in hematopoietic cells, and functions in the development and antigen-stimulated response of lymphocytes. Burkitt's lymphoma (BL) is characterized as transformed B cell lymphoma, and is highly associated with Epstein-Barr virus (EBV). EBV nuclear antigen 1 (EBNA1) is the only viral protein expressed across all three types of latency and essential for the persistence of EBV genome. It is not clear yet how EBNA1 contributes to the growth advantage of latently infected cells such as in EBV+ lymphoma B cells. Here, we reported that Vav1 interacts with EBNA1 via its C-terminal SH3 domain. This interaction suppresses the expression of a pro-apoptotic Bcl-2 family member, Bim, resulting in the resistance of the BL cells to apoptotic inductions. Our data uncovered Vav1 as a novel target for EBNA1, and suggested a pro-survival role of Vav1 in the pathogenesis of EBV associated BLs.

The ratio of Bcl-2/Bim as a predictor of cisplatin response provides a rational combination of ABT-263 with cisplatin or radiation in small cell lung cancer.

Cisplatin-based chemotherapy and radiotherapy are the most commonly used treatments for small cell lung cancer (SCLC). However, despise initially dramatic response, the response duration of SCLC patients is variable and resistance to chemo- and radio-therapy inevitably develops. The aim of the study is to investigate the role of Bcl-2 family proteins in predicting SCLC sensitivity to cisplatin treatment, and to identify the potential sensitizer of cisplatin or ratiation treatment in SCLC. We collected cisplatin sensitivity data from public available database, and evaluated its possible association with mRNA or protein expression of Bcl-2 family members in SCLC cell lines. The IC50 value of cisplatin was significantly correlated with the ratio of Bcl-2/Bim mRNA expression in 33 SCLC cell lines (P= 0.041) as well as the ratio of Bcl-2/Bim protein expression in 7 SCLC cell lines (P= 0.0252). Furthermore, a BH3-mimetic ABT-263 was found to be able to sensitize SCLC cells to cisplatin or radiation. The synergistic and additive antitumor activity of ABT-263 combined with cisplatin or radiation was associated with the enhanced apoptosis, which may be caused by the disruption of Bcl-2 binding to Bim by ABT-263. Our study indicates that the ratio of Bcl-2/Bim could be a SCLC response predictor to cisplatin, and ABT-263 addition could be an effective strategy to improve the activity of chemo- or radio-therapy in SCLC.

Downregulation of intrinsic apoptosis pathway in erythroblasts contributes to excessive erythrocytosis of chronic mountain sickness

Chronic mountain sickness (CMS) has a higher incidence in the plateau region and is characterized by excessive erythrocytosis and hypoxemia. Bcl-2 family plays an important role in the process of erythropoiesis and the regulation of apoptosis. This study aimed to examine the change in apoptosis of erythroblasts in CMS patients and explore the involvement of Bcl-2 family. Bone marrow mononuclear cells (BMMNCs) were isolated by density gradient centrifugation from 18 CMS patients and 17 control participants. The apoptotic rate, mitochondrial membrane potential (MMP), the protein expression of caspase-3, TNFR, Fas, Bcl-2, Bax and Cyt-C were examined by flow cytometry, and mRNA expression was determined by real-time PCR. The results showed that apoptotic rate of erythroblasts was lower and MMP was higher in CMS group than in control group. The mRNA and protein expression levels of Bcl-2 were higher while Bax level was lower in CMS group than in control group. In CMS group, the apoptosis rate of CD71+ erythroblasts was negatively correlated with the ratio of CD71+ cells in BMMNCs and positively correlated with hemoglobin level. In conclusion, erythroblasts apoptosis is decreased due to the regulation of the expression of Bcl-2 family members in the erythroblasts of CMS patients.

Molecular Effects of Treatment of Human Colorectal Cancer Cells with Natural and Classical Chemotherapeutic Drugs: Alterations in the Expression of Apoptosis-related BCL2 Family Members, Including BCL2L12.

Current chemotherapy regimens for the treatment of colorectal cancer (CRC) include oxaliplatin, irinotecan, and fluorouracil along with leucovorin. Cytotoxicity involves the induction of programmed cell death. The purpose of this study was to assess the molecular effects of doxorubicin (a 14-OH derivative of the natural product daunorubicin) and common chemotherapeutic drugs (used in the clinical practice to treat CRC) on the expression of the most prominent members of the BCL2 family, namely BCL2, BAX, BCLX, and MCL1. Moreover, we sought to define the role of BCL2L12, another member of the BCL2 family, the apoptotic role of which is ambiguous. The MTT cell proliferation assay was used to determine the IC50 of each chemotherapeutic drug at 72 hours of treatment of Caco-2 and DLD-1 colorectal adenocarcinoma cell lines. Real-time PCR was used to quantify the antiapoptotic BCL2-α, BLCX-L, and MCL1-L transcripts, the proapoptotic BAX, BLCX-S, BLCX-ES, MCL1-S, and MCL1-ES transcripts, and BCL2L12 expression in relation to GAPDH mRNA levels. We constructed growth curves of Caco-2 and DLD-1 cells and determined the IC50 of each drug at 72 hours of treatment. Significant alterations in the expression levels of the studied BCL2 family genes and/or particular transcripts were observed. The intrinsic apoptotic pathway is activated during treatment of CRC cells with common chemotherapeutic drugs. Moreover, BCL2L12 mRNA expression increases progressively during treatment, similarly to the expression of other BCL2 family genes favoring apoptosis and/or particular proapoptotic transcripts, thus suggesting a proapoptotic role for BCL2L12 in chemotherapy-treated CRC cells.

BCL2-Family Dysregulation in B-Cell Malignancies: From Gene Expression Regulation to a Targeted Therapy Biomarker.

BCL2-family proteins have a central role in the mitochondrial apoptosis machinery and their expression is known to be deregulated in many cancer types. Effort in the development of small molecules that selectively target anti-apoptotic members of this family i.e., Bcl-2, Bcl-xL, Mcl-1 recently opened novel therapeutic opportunities. Among these apoptosis-inducing agents, BH3-mimetics (i.e., venetoclax) led to promising preclinical and clinical activity in B cell malignancies. However, several mechanisms of intrinsic or acquired resistance have been described ex vivo therefore predictive markers of response as well as mechanism-based combinations have to be designed. In the present study, we analyzed the expression of the BCL2-family genes across 10 mature B cell malignancies through computational normalization of 21 publicly available Affimetrix datasets gathering 1,219 patient samples. To better understand the deregulation of anti- and pro-apoptotic members of the BCL2-family in hematological disorders, we first compared gene expression profiles of malignant B cells to their relative normal control (naïve B cell to plasma cells, n = 37). We further assessed BCL2-family expression according to tissue localization i.e., peripheral blood, bone marrow, and lymph node, molecular subgroups or disease status i.e., indolent to aggressive. Across all cancer types, we showed that anti-apoptotic genes are upregulated while pro-apoptotic genes are downregulated when compared to normal counterpart cells. Of interest, our analysis highlighted that, independently of the nature of malignant B cells, the pro-apoptotic BH3-only BCL2L11 and PMAIP1 are deeply repressed in tumor niches, suggesting a central role of the microenvironment in their regulation. In addition, we showed selective modulations across molecular subgroups and showed that the BCL2-family expression profile was related to tumor aggressiveness. Finally, by integrating recent data on venetoclax-monotherapy clinical activity with the expression of BCL2-family members involved in the venetoclax response, we determined that the ratio (BCL2+BCL2L11+BAX)/BCL2L1 was the strongest predictor of venetoclax response for mature B cell malignancies in vivo.

A Novel MCL1 Inhibitor Combined with Venetoclax Rescues Venetoclax-Resistant Acute Myelogenous Leukemia.

Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 association, leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addition, VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clinical trials. SIGNIFICANCE: Targeting antiapoptotic proteins in AML is a key therapeutic strategy, and MCL1 is a critical antiapoptotic oncoprotein. Armed with novel MCL1 inhibitors and the potent BCL2 inhibitor venetoclax, it may be possible to selectively induce apoptosis by combining or thoughtfully sequencing these inhibitors based on a rational evaluation of AML.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

Curcumin Attenuates Testicular Injury in Rats with Streptozotocin-Induced Diabetes.

Oxidative damage, inflammation, and apoptosis are the primary features of diabetic testicular damage. Curcumin protects against diabetic testicular injury, but the underlying mechanisms remain obscure. This study examined the effect of curcumin on type 2 diabetes mellitus- (T2DM-) induced testicular injury, oxidative stress, and apoptotic changes. T2DM rats were intraperitoneally injected with 40 mg/kg STZ after being fed a high-fat diet for 8 weeks. One week after STZ injection, 100 or 200 mg/kg curcumin was administered orally to the diabetic rats for 16 weeks. Histological changes in the testes were determined by HE staining. Serum testosterone was measured. Markers of superoxide levels, such as SOD activity and MDA content, and markers of cell death, including the expression of Bax, Bcl-2, and MAPK family members, were measured by molecular biology or immunohistochemical techniques. Degeneration and disruption of seminiferous tubule structure were observed in diabetic rats. Serum testosterone levels were markedly lower in diabetic rats than in control rats. Moreover, testicular apoptosis and Bax expression were much higher in diabetic rats than in control rats. Superoxide generation, the NADP+/NADPH ratio, and NADPH oxidase subunit expression, including expression of the gp91phox, p47phox, and p67phox subunits, increased, while antioxidant enzyme levels decreased in diabetic rats. Furthermore, the MAPK signaling pathway was activated in diabetic rats. Curcumin partially prevented diabetes-induced microstructural abnormalities and significantly increased serum testosterone levels compared to untreated T2DM rats. Additionally, curcumin reduced testicular apoptosis by regulating apoptotic proteins and markedly inhibited oxidative stress levels by downregulating MDA expression, decreasing NADPH activity, and restoring antioxidant enzymes. Remarkably, curcumin treatment also suppressed MAPK activation. Thus, curcumin may have therapeutic value in the treatment of diabetes-induced testicular injury due to its prevention of testicular apoptosis and attenuation of oxidative stress.

Abstract 880: Anti-tubulin antibody drug conjugates potentiate venetoclax activity in non-Hodgkin lymphoma by targeting MCL-1

Abstract Venetoclax, a potent, orally bioavailable inhibitor that selectively targets BCL-2 and induces intrinsic apoptosis, is currently approved for the treatment of relapsed/refractory chronic lymphocytic leukemia with 17p deletion (R/R 17p del CLL) and also has profound efficacy in broad R/R CLL as a single agent or in combination with obinutuzumab (anti-CD20 antibody). However, evidence of robust efficacy by venetoclax as a single agent in certain subtypes of non-Hodgkin lymphoma (NHL) has been limited. For example, diffuse large B-Cell lymphoma (DLBCL) is the most common sub-type of NHL, yet the overall response rate of DLBCL patients to venetoclax was 18% in contrast to 61% in other NHL sub-types. It has been proposed that expression of other pro-survival BCL-2 family members may limit single-agent venetoclax efficacy and is thus considered a potential major resistance factor. To this end, we have observed that low sensitivity to venetoclax monotherapy is correlated with MCL-1 expression in NHL cell lines. We have previously reported that anti-tubulin agents promote MCL-1 degradation. Given that polatuzumab vedotin is a novel antibody drug conjugate targeted to CD79b and delivers an auristatin anti-tubulin agent, we hypothesized that combining polatuzumab with venetoclax presents a mechanism-driven combination strategy for maximizing anti-tumor responses in NHL. In NHL patients, both polatuzumab vedotin and venetoclax have acceptable and non-overlapping toxicities and the combination of both drugs may provide an improved therapeutic index relative to conventional chemotherapy. Here, we demonstrate that polatuzumab vedotin synergizes with venetoclax both in vitro and in vivo. Mechanistically, the combination of both drugs promotes apoptosis in NHL cell lines as indicated by caspase 3/7 activation and dependence on BAX/BAK expression, and decreases MCL-1 protein levels. Furthermore, the combination of venetoclax with polatuzumab vedotin is more efficacious in vitro than the combination of venetoclax with a selective MCL-1 small molecule inhibitor (AMG 176). The combination of polatuzumab vedotin with venetoclax results in durable tumor regressions in diffuse large B-cell and mantle cell lymphoma xenograft models at tolerated doses and is more efficacious than the combination of venetoclax plus bendamustine, a standard chemotherapeutic agent. Based on our pre-clinical data and the strong mechanistic rationale, a drug combination regimen of venetoclax with polatuzumab vedotin and obinutuzumab is currently being evaluated in Phase1b clinical trials in R/R FL and DLBCL (ClinicalTrials.gov identifier NCT02611323). Citation Format: Dhara N. Amin, Jason Oeh, Anuradha Zindal, Lisa Musick, Jamie Hirata, Mehrdad Mobasher, Andy Polson, Deepak Sampath, Ingrid E. Wertz. Anti-tubulin antibody drug conjugates potentiate venetoclax activity in non-Hodgkin lymphoma by targeting MCL-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 880.

P53/PUMA are potential targets that mediate the protection of brain-derived neurotrophic factor (BDNF)/TrkB from etoposide-induced cell death in neuroblastoma (NB).

The over-expressions of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB have been reported to induce chemo-resistance in neuroblastoma (NB) cells. In this study, we investigated the roles of P53 and BCL2 family members in the protection of BDNF/TrkB from etoposide-induced NB cell death. TB3 and TB8, two tetracycline (TET)-regulated TrkB-expressing NB cell lines, were utilized. The expressions of P53 and BCL2 family members were detected by Western blot or RT-PCR. Transfection of siRNAs was used to knockdown P53 or PUMA. Activated lentiviral was used to over-express PUMA. Cell survival was performed by MTS assay, and the percentage of cell confluence was measured by IncuCyte ZOOM. Our results showed that etoposide treatment induced significant and time-dependent increase of P53, which could be blocked by pre-treatment with BDNF, and knockdown P53 by transfecting siRNA attenuated etoposide-induced TrkB-expressing NB cell death. PUMA was the most significantly changed BCL2 family member after treatment with etoposide, and pre-treatment with BDNF blocked the increased expression of PUMA. Transfection with siRNA inhibited etoposide-induced increased expression of PUMA, and attenuated etoposide-induced NB cell death. We also found that over-expression of PUMA by infection of activated lentiviral induced TrkB-expressing NB cell death in the absence of etoposide, and treatment of BDNF protected NB cells from PUMA-induced cell death. Our results suggested that P53 and PUMA may be potential targets that mediated the protection of BDNF/TrkB from etoposide-induced NB cell death.