Upregulation Of Anti-apoptotic Proteins Research Articles

Abstract 3277: PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer

Abstract [Purpose] The mesenchymal epithelial transition factor receptor (MET) is a potential therapeutic target in various cancers, including non-small cell lung cancer (NSCLC). In NSCLC, the activation of MET pathway is thought to occur through diverse mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. These tumors show oncogene addiction to MET and exhibit remarkable responses to MET-Tyrosine Kinase Inhibitors (TKIs). However, the long-term effectiveness of MET-TKIs is usually limited due to acquired drug resistance. The mechanisms underlying primary and acquired resistance to MET-TKIs in the management of NSCLC remain unclear. In this study, we investigated the mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. [Experimental Designs] Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. The factors contributing to the resistance mechanism were assessed by genomic and transcriptomic data, and the alterations hypothesized to confer resistance were validated. [Results] Multiple mechanisms underlay acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the drug resistance mechanism was mediated by the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1). Crizotinib resistance was overcome by combination therapy with PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial to mesenchymal transition (EMT) with the upregulation of antiapoptotic proteins. In the H1993-derived resistant cells, the phosphorylated forms of MEK and ERK were observed to be upregulated and MEK inhibitors could be a potential therapeutic strategy for overcoming the resistance. [Conclusions] In this study, we revealed the different mechanisms of drug resistance to MET-TKI treatment and presented some possible strategies to overcome the resistance. Citation Format: Tomoaki Higashihara, Yin Min Thu, Ken Suzawa, Keiichi Date, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Naohiro Hayashi, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3277.

Uncovering the Therapeutic Potential of Phosphocreatine in Diabetic Retinopathy: Mitigating Mitochondrial Dysfunction and Apoptosis via JAK2/STAT3 Signaling Pathway.

Diabetic retinopathy (DR) stands as a prevalent complication of diabetes mellitus, causing damage to the delicate retinal capillaries and potentially leading to visual impairment. While the exact underlying cause of DR remains elusive, compelling research suggests that mitochondrial energy deficiency and the excessive generation of reactive oxygen species (ROS) play pivotal roles in its pathogenesis. Recognizing that controlling hyperglycemia alone fails to reverse the defects in retinal mitochondria induced by diabetes, current strategies seek to restore mitochondrial function as a means of safeguarding against DR. To address this pressing issue, a comprehensive study was undertaken to explore the potential of phosphocreatine (PCr) in bolstering mitochondrial bioenergetics and providing protection against DR via modulation of the JAK2/STAT3 signaling pathway. Employing rat mitochondria and RGC-5 cells, the investigation meticulously assessed the impact of PCr on ROS production, mitochondrial membrane potential, as well as the expression of crucial apoptotic and JAK2/STAT3 signaling pathway proteins, utilizing cutting-edge techniques such as high-resolution respirometry and western blotting. The remarkable outcomes revealed that PCr exerts a profound protective influence against DR by enhancing mitochondrial function and alleviating diabetes-associated symptoms and biochemical markers. Notably, PCr administration resulted in an upregulation of antiapoptotic proteins, concomitant with a downregulation of proapoptotic proteins and the JAK2/STAT3 signaling pathway. These significant findings firmly establish PCr as a potential therapeutic avenue for combating diabetic retinopathy. By augmenting mitochondrial function and exerting antiapoptotic effects via the JAK2/STAT3 signaling pathway, PCr demonstrates promising efficacy both in vivo and in vitro, particularly in counteracting the oxidative stress engendered by hyperglycemia. In summary, our study sheds light on the potential of PCr as an innovative therapeutic strategy for diabetic retinopathy. By bolstering mitochondrial function and exerting protective effects via the modulation of the JAK2/STAT3 signaling pathway, PCr holds immense promise in ameliorating the impact of DR in the face of oxidative stress induced by hyperglycemia.

PAI-1 mediates acquired resistance to MET-targeted therapy in non-small cell lung cancer.

Mechanisms underlying primary and acquired resistance to MET tyrosine kinase inhibitors (TKIs) in managing non-small cell lung cancer remain unclear. In this study, we investigated the possible mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. Genomic and transcriptomic data were used to assess the factors contributing to the resistance mechanism, and the alterations hypothesized to confer resistance were validated. Multiple mechanisms underlie acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1), mediated the drug resistance mechanism. Crizotinib resistance was addressed by combination therapy with a PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial-to-mesenchymal transition with the upregulation of antiapoptotic proteins. In H1993-derived resistant cells, MEK inhibitors could be a potential therapeutic strategy for overcoming resistance with downstream mitogen-activated protein kinase pathway activation. In this study, we revealed the different mechanisms of acquired resistance to the MET inhibitor crizotinib with potential therapeutic application in patients with MET-amplified lung carcinoma.

Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state

Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state

Thymosin β4 Exerts a Cytoprotective Function and Attenuates Liver Injury in Murine Hepatic Sinusoidal Obstruction Syndrome after Hematopoietic Stem Cell Transplantation

Hepatic sinusoidal obstruction syndrome (HSOS) is a life-threatening complication that may occur after hematopoietic stem cell transplantation (HSCT). Hepatic sinusoidal endothelial cell (HSEC) injury and liver fibrosis are key mechanisms of HSOS. Thymosin β4 (Tβ4) is an active polypeptide that functions in a variety of pathologic and physiologic states, including inflammation regulation, anti-apoptosis, and anti-fibrosis. In this study, we found that Tβ4 can stimulate HSEC proliferation, migration, and tube formation in vitro via activation of pro-survival signaling AKT (protein kinase B). In addition, Tβ4 resisted γ irradiation-induced HSEC growth arrest and apoptosis in parallel with upregulation of anti-apoptotic protein B cell lymphoma extra-large (Bcl-xL) and B cell lymphoma-2 (Bcl-2), which may be associated with activation of AKT. More importantly, Tβ4 significantly inhibited irradiation-induced pro-inflammatory cytokines in parallel with negative regulation of TLR4/MyD88/NF-κB and MAPK p38. Meanwhile, Tβ4 reduced intracellular reactive oxygen species production and upregulated antioxidants in HSECs. Additionally, Tβ4 inhibited irradiation-induced activation of hepatic stellate cells by downregulating the expression of fibrogenic markers α-SMA, PAI-1, and TGF-β. In a murine HSOS model, levels of circulating alanine aminotransferase, aspartate aminotransferase, total bilirubin, and pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly reduced after administration of Tβ4 peptide; furthermore, Tβ4 treatment successfully ameliorated HSEC injury, inflammatory damage, and fibrosis of the murine liver. Taken together, our findings indicate that Tβ4 stimulates proliferation and angiogenesis of HSECs, exerts a cytoprotective effect, and attenuates liver injury in a murine HSOS model, suggesting that its use may be a potential strategy to prevent and treat HSOS after HSCT.

Involvement of Bcl-2 Family Proteins in Tetraploidization-Related Senescence.

The B-cell lymphoma 2 (Bcl-2) family of proteins is the main regulator of apoptosis. However, multiple emerging evidence has revealed that Bcl-2 family proteins are also involved in cellular senescence. On the one hand, the different expression of these proteins determines the entry into senescence. On the other hand, entry into senescence modulates the expression of these proteins, generally conferring resistance to apoptosis. With some exceptions, senescent cells are characterized by the upregulation of antiapoptotic proteins and downregulation of proapoptotic proteins. Under physiological conditions, freshly formed tetraploid cells die by apoptosis due to the tetraploidy checkpoint. However, suppression of Bcl-2 associated x protein (Bax), as well as overexpression of Bcl-2, favors the appearance and survival of tetraploid cells. Furthermore, it is noteworthy that our laboratory has shown that the joint absence of Bax and Bcl-2 antagonist/killer (Bak) favors the entry into senescence of tetraploid cells. Certain microtubule inhibitory chemotherapies, such as taxanes and vinca alkaloids, induce the generation of tetraploid cells. Moreover, the combined use of inhibitors of antiapoptotic proteins of the Bcl-2 family with microtubule inhibitors increases their efficacy. In this review, we aim to shed light on the involvement of the Bcl-2 family of proteins in the senescence program activated after tetraploidization and the possibility of using this knowledge to create a new therapeutic strategy targeting cancer cells.

Immune Phenotypic Characterization of a TRAIL-Knockout Mouse

The TNF-superfamily member TRAIL is known to mediate selective apoptosis in tumor cells suggesting this protein as a potential antitumor drug target. However, initial successful pr-clinical results could not be translated into the clinic. Reasons for the ineffectiveness of TRAIL-targeting in tumor therapies could include acquired TRAIL resistance. A tumor cell acquires TRAIL resistance, for example, by upregulation of antiapoptotic proteins. In addition, TRAIL can also influence the immune system and thus, tumor growth. We were able to show in our previous work that TRAIL-/- mice show improved survival in a mouse model of pancreatic carcinoma. Therefore, in this study we aimed to immunologically characterize the TRAIL-/- mice. We observed no significant differences in the distribution of CD3+, CD4+, CD8+ T-cells, Tregs, and central memory CD4+ and CD8+ cells. However, we provide evidence for relevant differences in the distribution of effector memory T-cells and CD8+CD122+ cells but also in dendritic cells. Our findings suggest that T-lymphocytes of TRAIL-/- mice proliferate at a lower rate, and that the administration of recombinant TRAIL significantly increases their proliferation, while regulatory T-cells (Tregs) from TRAIL-/- mice are less suppressive. Regarding the dendritic cells, we found more type-2 conventional dendritic cells (DC2s) in the TRAIL-/- mice. For the first time (to the best of our knowledge), we provide a comprehensive characterization of the immunological landscape of TRAIL-deficient mice. This will establish an experimental basis for future investigations of TRAIL-mediated immunology.

Sericin improves memory and sociability impairments evoked by transient global cerebral ischemia through suppression of hippocampal oxidative stress, inflammation, and apoptosis.

Sericin (Ser) is a natural neuroactive macromolecule with diverse pharmacological properties, and our previous findings have shown its neuroprotective potentials. This study aimed to investigate the therapeutic potential of Ser on cognitive dysfunction induced by transient global cerebral ischemia/reperfusion (tGI/R) and its mechanism of action. The tGI/R was induced in BALB/c mice by bilateral occlusion of the common carotid arteries for two 5 min followed by a 10-min reperfusion period. After 24 h, mice were treated with normal saline or different doses of Ser (100, 200, and 300 mg/kg) for 10 days. Cognitive performances were assessed using the Barnes maze and social interaction tasks. Oxidative stress markers including superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant capacity (TAC), and malondialdehyde (MDA) as well as pro-inflammatory cytokines (interleukin (IL)-6 and tumor necrosis factor-alpha) and anti-inflammatory cytokine (IL-10) were assessed in the hippocampus. Markers of apoptosis (pro- and cleaved caspase-9 and 3, Bax, and Bcl-2) were assessed by Western blotting. Besides, transferase-mediated dUTP nick end-labeling assay was used to detect apoptotic cell death. We show here that Ser administration improved tGI/R-induced cognitive deficits, enhanced the activity of SOD and GPx, increased TAC levels, while reduced MDA levels. Notably, Ser decreased neuronal apoptotic cell death in the hippocampal dentate gyrus (DG) region, accompanied by suppression of neuroinflammation, downregulation of pro-apoptotic proteins (caspase-9, caspases-3, and Bax), and upregulation of anti-apoptotic protein, Bcl-2. Taken together, Ser administration protected hippocampal neurons from apoptotic cell death by impeding oxidative stress and inflammatory responses and, in turn, improved cognitive function in the tGI/R mice.

Nanoliposomal C6-Ceramide Exhibits Synergistic Anti-Cancer Activity with a Small Molecule Agonist of PP2A in Chronic Lymphocytic Leukemia

The introduction of targeted therapies, including venetoclax (inhibitor of Bcl-2) and ibrutinib (inhibitor of Bruton's tyrosine kinase), has significantly advanced treatment of non-Hodgkin's lymphoma, particularly Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, patients treated with ibrutinib, venetoclax, or the combination of ibrutinib and venetoclax are likely to exhibit treatment relapse. We have previously published that microenvironmental factors can generate anti-apoptotic resistance to combinatorial ibrutinib/venetoclax in both CLL and MCL models via NF-kB-dependent upregulation of anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. 2017). We have recently published the discovery of phenotypically similar drug resistant CLL cells that overexpress multiple anti-apoptotic proteins in patients de novo. These cells were enriched during treatment with venetoclax, suggesting this pool of resistant persister cells could be a source of treatment failure (Jayappa et al. 2021). We have previously formulated a non-toxic C6-ceramide nanoliposome (CNL) that greatly improves bioavailability of this bioactive sphingolipid and exerts anti-cancer activity in vivo, including CLL cell line that exhibits multidrug resistance phenotype (Lindsay et al. 2013). Thus, CNL-based novel therapies could hold promise for depleting the pool of multidrug resistant cancer cells in patients. In the current study, we screened CNL activity in CLL patient PBMC samples (N=15) using a flow cytometry-based cytotoxicity assay ex vivo. Our analysis revealed that CNL induced apoptotic/non-apoptotic cell death at overly high concentrations (EC50, 31 µM), prompting us to search for potential combinatorial therapies with enhanced toxicity profiles. To address this, we performed a combination drug screen with CNL as an anchor agent in a multidrug resistant CLL cell line (MEC1, p53-/mt) using a cell survival assay. Our analysis discovered exceptional synergistic interaction between CNL and a small molecule activator of Protein Phosphatase 2A (PP2A) (SMAP, DT061) in MEC1 cells [Bliss score (actual-Bliss predicted cytotoxicity): 56% (CNL, 6 µM; DT061, 12 µM)] and this synergistic behavior was further validated in primary CLL samples (N=5) ex vivo. Analyses using patient PBMC samples (N=3) have shown that the CNL and SMAP combination triggers apoptotic cell death with cleavage of caspase9, capase3, and PARP in CLL cells, while normal T lymphocytes in these samples were relatively less affected. At the molecular level, mutually reinforcing interactions between SMAP and CNL were observed. Using mass-spectrometric profiling of sphingolipids, we discovered that PP2A activation by SMAP modulates sphingolipid metabolism, leading to enhanced accumulation of C6-ceramide as well as synthesis of endogenous, pro-apoptotic sphingolipid molecules (C14- and C16-ceramides) in cells exposed to CNL. In addition, co-treatment with SMAP and CNL induced synergistic/additive downregulation of pBAD and cMyc, suggesting cMyc and/or Bad-dependent pathways could serve as node of convergence for synergistic interaction seen in these cells. In summary, we demonstrate that the CNL-SMAP combination displays exceptional synergy in CLL cells. At the molecular level, CNL and SMAP exhibit mutually reinforcing interactions that potentially converge on cMyc and Bad pathways, leading to synergistically increased apoptotic death in these cells. We intend to further characterize the role of cMyc and/or Bad pathway in synergistic interaction and assess the in vivo safety/efficacy of CNL-SMAP combination using a CLL xenograft mouse model. Collectively, this work provides the basis for further testing of this combination in in vivo models.

Abstract 4174: Augmenting NK cell based immunotherapy by targeting mitochondrial apoptosis

Abstract Theoretically a broad spectrum of cancers, including those with low tumor burdens, could respond to NK cell therapy and NK cells might combat resistance to T cell-based therapies. Allogenic NK cells have also demonstrated a good safety profile in clinical trials. These promising features have led to increasing efforts to advance NK-based immunotherapies in recent years. However, efficacy of NK therapy remains limited. Strategies to augment NK efficacy are therefore much needed. To augment the efficacy of adoptive NK cells, most current studies revolve around two focal points: optimizing the source of NK cells and improving their functionality and persistence in vivo. In the current study, we took a different approach by studying how to make cancer cells more vulnerable to NK-mediated killing. It remains unclear to what extent mitochondrial apoptosis is required for NK-mediated killing. We found that primary human NK cells robustly induce mitochondrial apoptosis. Moreover, mitochondrial apoptosis is essential for efficient NK killing, especially at physiologically relevant low E:T ratios (Effector:Target ratios). It is traditionally believed that cytotoxic cell-cancer cell contacts are binary live/death events. We found that NK engagement is often sub-lethal and push cancer cells towards apoptotic threshold (i.e. priming cancer cells for apoptosis), making them more susceptible for killing by subsequent NK contacts. Upregulation of anti-apoptotic proteins has been widely implicated in cancer resistance to chemo- and targeted therapies. We found that overexpression of these proteins such as BCL-2, BCL-XL, and MCL-1 reduced mitochondrial priming for apoptosis and made cancer cells less susceptible to NK killing. We reasoned that additional agents that increase cancer cell mitochondrial priming for apoptosis (e.g., BH3 mimetics) might augment NK-induced killing, as long as NK cells can tolerate these agents. While primary resting NK cells are sensitive to BCL-2, BCL-XL, and MCL-1 inhibitors, unexpectedly, pre-activation with IL-2 conferred resistance of NK cells to these inhibitors. NK cells and BH3 mimetics synergized in both priming and killing cancer cells in vitro. BH3 profiling could also predict the ideal BH3 mimetics to be combined with NK cells for different tumor models. Using liquid and solid tumor xenograft models, we demonstrated that BH3 mimetics synergized with NK cells in suppressing tumor growth and prolonging mouse survival. In summary, we propose a rational strategy to sensitize cancer cells to NK cellular therapy. Moreover, we elucidate the mechanism underlying the apoptotic signaling that is the scientific basis for this strategy. Our results could potentially enable basic, pre-clinical, and clinical studies investigating the combined effects of BH3 mimetics with NK cells in cancers. Citation Format: Rongqing Aaron Pan, Jeremy Ryan, Deng Pan, Kai Wucherpfennig, Anthony Letai. Augmenting NK cell based immunotherapy by targeting mitochondrial apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4174.

Abstract 5652: A novel anti apoptotic function of CTLA4 in melanoma

Abstract Cytotoxic T Lymphocyte Antigen 4 (CTLA4) is an important immune checkpoint protein and has been utilized as an immunotherapeutic target against melanoma. Our previous studies have shown that CTLA4 is expressed at low levels in primary human melanocytes but is upregulated in mutant-BRAF/NRAS melanoma cells and tissues. Despite predominantly intracellular localization of CTLA4, its intracellular function remains highly contradictory and unsubstantiated. We found that ectopic expression of Ctla4 in mouse melanoma cell lines substantially promoted lung colonization in allograft model systems in syngeneic mice. Moreover, similar results were observed in immunocompromised recipient mice, suggesting an intracellular cell-autonomous tumorigenic role of Ctla4 in melanoma. These finding led us to investigated intracellular functions of CTLA4 and its potential roles in melanomagenesis. Through the Ingenuity Pathway Analysis of our proteomics data, we found apoptosis as a major affected pathway in Ctla4-expressing mouse melanoma cells. We treated the cells (B2905-Ctla4-ee and EV control) with doxorubicin to induce apoptosis, followed by assessment by Annexin V assay. We found that Ctla4 expressing mouse melanoma cells were significantly resistant to doxorubicin-induced apoptosis. In reciprocal experiment, we generated (by CRISPR-Cas9) CTLA4-knockout A2058 human melanoma cells that exhibit high endogenous CTLA4 expression and we found that knockout cells showed significantly increased apoptosis than the parental cells. Moreover, we also observed significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) in CTLA4-expressing human melanoma cell lines. CTLA4-expressing cells also showed significantly higher invasion in Matrigel-coated transwell assay. These findings lead us to conclude that CTLA4 plays a significant role in regulating apoptosis and promoting melanoma progression in BRAF/NRAS mutant cells. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. A novel anti apoptotic function of CTLA4 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5652.

Targeting metabolism to overcome cancer drug resistance: A promising therapeutic strategy for diffuse large B cell lymphoma.

Cancer cell metabolism including aerobic glycolysis, amino acid and fatty acid metabolism, has been extensively studied. Metabolic reprogramming is a major hallmark of cancer, which promotes cancer cell proliferation, progression and metastasis, as well as provokes resistance to chemotherapeutic drugs. Several signal transduction pathways, such as BCR, MEK/ERK, Notch, NF-κB and PI3K/AKT/mTOR, regulate tumor metabolism, hence promoting tumor cell growth, proliferation and progression. Therefore, targeting metabolic enzymes, metabolites or their signal transduction pathways may constitute a promising therapeutic strategy to enhance cancer treatment efficacy. Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of non-Hodgkin lymphoma (NHL), and one-third of DLBCL patients suffer from relapsed/refractory disease after chemotherapy. The mechanisms underlying drug resistance are complex, including target gene mutations, metabolic reprogramming, aberrant signal transduction pathways, enhanced drug efflux via overexpression of multidrug efflux transporters like P-glycoprotein, upregulation of anti-apoptotic proteins, drug sequestration and enhanced DND repair. This review delineates the distinct metabolic reprogramming patterns and the association between metabolism and anticancer drug resistance in DLBCL as well as the emerging strategies to surmount chemoresistance in DLBCL.

ROS-mediated activation of p38 protects hepatocellular carcinoma cells from caspase-independent death elicited by lysosomal damage

Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancer in the world. Despite this, few effective drugs are available for its treatment, in part due to the development of resistance, and surgical resection remains the most valuable option, when applicable. Upregulation of anti-apoptotic proteins, downregulation of pro-apoptotic factors and the acquisition of mutations in signaling pathways leading to caspase activation are a few examples of mechanisms that allow cancer cells to evade caspase-dependent apoptosis and continue to grow. The identification of drugs triggering the activation of caspase-independent death may therefore be an effective strategy to circumvent resistance and kill cancer cells. Here, we show that the lysosome damaging compound glycyl-l-phenylalanine 2-naphthylamide (GPN) induces cell death by a caspase-independent mechanism in HCC cell lines. Additionally, we identify the MAPK p38 as a novel mediator of the lysosomal stress response. Indeed, a ROS-dependent activation of p38 occurs in response to lysosomal damage, promoting the recovery of lysosomal integrity. As a consequence, pharmacological or genetic inhibition of p38 increases cell death elicited by GPN. Our findings identify p38 as a potential target to potentiate the cytotoxic effects of lysosomal damage and induce caspase-independent cell death in HCC cells, laying the ground for future evaluation of the efficacy of combination therapy.

Serum B-cell maturation antigen level in multiple myeloma

Background Multiple myeloma (MM) is a malignant neoplasm of plasma cells that accumulate in the bone marrow producing excessive amounts of monoclonal immunoglobulins (Ig) and suppression of other normal polyclonal Igs, leading to bone destruction and marrow failure. B-cell maturation antigen (BCMA) is known to bind to B-cell-activating factor (BAFF) or a proliferation-inducing ligand (APRIL), leading to the activation of cell proliferation pathways and upregulation of antiapoptotic proteins, which is considered the main step in the pathogenesis of MM. Aim This study was planned to evaluate serum BCMA levels in MM patients and its clinical significance. Patients and methods This study was carried out on 40 newly diagnosed MM patients and 40 apparently healthy individuals served as the control. The two studied groups were compared as regards serum BCMA levels using the enzyme-linked immunosorbent assay technique. Results Results showed that there was a statistically significant increase in MM patients when compared with the control group, and there was a significant positive correlation between BCMA level and plasma cells in bone marrow, M protein, number of bone lesions, and beta-2 microglobulin. However there was no correlation between BCMA level and age, creatinine, and hemoglobin. Conclusion Serum BCMA level was significantly elevated in MM patients compared with healthy controls that may suggest the role of BCMA in the pathogenesis of MM. The high sensitivity of BCMA suggests that it may be a novel diagnostic marker, and its level was positively correlated with other prognostic markers. It may be used as a prognostic marker in MM patients.

Bone Marrow Mesenchymal Stromal Cell-mediated Resistance in Multiple Myeloma Against NK Cells can be Overcome by Introduction of CD38-CAR or TRAIL-variant.

We have recently shown the strong negative impact of multiple myeloma (MM)-bone marrow mesenchymal stromal cell (BMMSC) interactions to several immunotherapeutic strategies including conventional T cells, chimeric antigen receptor (CAR) T cells, and daratumumab-redirected NK cells. This BMMSC-mediated immune resistance via the upregulation of antiapoptotic proteins in MM cells was mainly observed for moderately cytotoxic modalities. Here, we set out to assess the hypothesis that this distinct mode of immune evasion can be overcome by improving the overall efficacy of immune effector cells. Using an in vitro model, we aimed to improve the cytotoxic potential of KHYG-1 NK cells toward MM cells by the introduction of a CD38-specific CAR and a DR5-specific, optimized TRAIL-variant. Similar to what have been observed for T cells and moderately lytic CAR T cells, the cytolytic efficacy of unmodified KHYG-1 cells as well as of conventional, DR5-agonistic antibodies were strongly reduced in the presence of BMMSCs. Consistent with our earlier findings, the BMMSCs protected MM cells against KHYG-1 and DR5-agonistic antibodies by inducing resistance mechanisms that were largely abrogated by the small molecule FL118, an inhibitor of multiple antiapoptotic proteins including Survivin, Mcl-1, and XIAP. Importantly, the BMMSC-mediated immune resistance was also significantly diminished by engineering KHYG-1 cells to express the CD38-CAR or the TRAIL-variant. These results emphasize the critical effects of microenvironment-mediated immune resistance on the efficacy of immunotherapy and underscores that this mode of immune escape can be tackled by inhibition of key antiapoptotic molecules or by increasing the overall efficacy of immune killer cells.

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.

Modulation of the Tumor Suppressor Protein PP2A Using a Small Molecule Agonist Overcomes Multi-Drug Resistance through Mitochondrial Permeability Transition Pore (MPTP) Dependent Induction of Apoptosis in Chronic Lymphocytic Leukemia

The advent of molecularly targeted therapies has revolutionized the treatment of Non-Hodgkin's Lymphoma (NHL), including Chronic Lymphocytic Leukemia (CLL). Venetoclax (VEN, an inhibitor of Bcl-2) and ibrutinib (IBR, an inhibitor of BTK) generated excellent clinical responses in CLL patients singly and even more effectively in combination (Portell et al. Blood, 2019; Tam et al.NEJM, 2018). Despite its efficacy, the majority of VEN or VEN+IBR responses are partial and resistance usually develops. We previously reported that treatment with microenvironmental agonists ex vivo can generate anti-apoptotic resistance to the VEN+IBR combination in CLL cells via NF-kB-dependent upregulation of multiple anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. Blood Adv, 2017). Here, we report that circulating CLL cells of lymph node origin (CD69positive) exhibit resistance to several BH-domain antagonists (inhibitors of Bcl-2, Mcl-1, and Bcl-xL) when used as single agents. Apoptosis resistance was shown to occur at a pre-mitochondrial level by insufficient activation of Bax/Bak proteins (Fig. 1A-B). Supportive of these findings, ex vivo treatment of primary CLL cells with agonists (sCD40L, TLR9 agonist CpG-ODN, and IL10) known to be expressed in the cancer microenvironment in vivoalso resulted in apoptosis restriction due to defective Bax/Bak activation. Our molecular studies suggest that this resistance is driven by the upregulation of anti-apoptotic proteins, which generates an intracellular environment in which single-drug treatments allow pro-apoptotic proteins (e.g. Bim) to swap between upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2), leading to defective Bax/Bak activation. Hence, therapies aimed at depleting this reservoir must block multiple anti-apoptotic proteins simultaneously or bypass Bax/Bak-dependent apoptosis. Several cancers, including NHL, have evolved mechanisms to suppress the activity of Protein Phosphatase 2A (PP2A), a serine/threonine phosphatase known to regulate cell survival/proliferation. In our current study involving various cancer cell lines (~250), a small molecule agonist of PP2A (SMAP, TRC-382) showed broad activity across blood cancer cell lines. A further pharmacologically optimized SMAP compound (DT061), also known to be safe in animal models (Tohmé et al. JCI Insight, 2019), was effective even in blood cancer cell lines and agonist treated CLL samples resistant to several BH-domain antagonists (inhibitors on Mcl-1, Bcl-xL, and Bcl-2), suggesting that PP2A activation could overcome pre-mitochondrial apoptosis restriction. DT061 was able to overcome drug resistance in patient-derived CD69positive CLL cells through the induction of Bax/Bak-independent apoptosis (Fig. 1C). Supportive of this finding, DT061 was also able to induce apoptosis in Bax/Bak double knockout CLL cell line (MEC1) (Fig. 1D). To determine the mechanisms underlying SMAP-induced apoptosis, we examined additional pathways capable of triggering apoptosis such as mitochondrial permeability transition pore (MPTP), calcium channels, and VDAC1 using selected small molecule inhibitors. Only inhibitors blocking MPTP (NIM811 or cyclopsorin-A/CspA) significantly impaired the DT061- but not VEN-induced apoptosis in primary CLL cells (Fig. 2A-B). Additionally, our analysis using the CalceinAM/CoCl2 method revealed that DT061 was able to induce MPTP opening in primary CLL cells, which was inhibited in the presence of NIM811 or CspA, suggesting that DT061 induces apoptosis through MPTP activation (Fig. 2C). In summary, we identify, in the circulation of treatment-naïve and treated CLL patients, microenvironmentally-activated leukemic B cells that exhibit apoptosis resistance at the level of Bax/Bak activation. Therapies aimed at depleting this reservoir of drug-resistant leukemic B cells must trigger a process of Bax/Bak independent apoptosis. We demonstrate that PP2A activation using the SMAP (DT061) induces apoptosis in drug resistant CLL cells bypassing the Bax/Bak pathway. The apoptosis induction was dependent on MPTP activation. Collectively, this work highlights the existence of an anti-apoptotic multi-drug resistant pool of CLL cells in patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir. Disclosures Williams: Janssen: Research Funding; Pharmacyclics: Research Funding; TG Therapuetics: Research Funding; Celgene: Honoraria; Gilead: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Kite: Honoraria; Xian Janssen: Honoraria. Portell:Xencor: Research Funding; Roche/Genentech: Consultancy, Research Funding; Infinity: Research Funding; TG Therapeutics: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding. Narla:The Icahn School of Medicine at Mount Sinai: Patents & Royalties: nternational Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2; Mount Sinai: Other: Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology.; RAPPTA Therapeutics: Consultancy, Current equity holder in private company.

Inhibiting the inhibitors: Targeting anti-apoptotic proteins in cancer and therapy resistance.

The cytotoxic effect of anti-cancer drugs relies on their ability to induce programmed cell death known as apoptosis. Evading apoptosis is a common characteristic of cancer cells and it is linked to both carcinogenesis and anticancer drug resistance. To escape apoptosis, cancer cells often express high levels of anti-apoptotic proteins and become "addicted "to them for their survival. Consequently, anti-apoptotic proteins have emerged as attractive druggable targets for the development of cancer therapeutics. In this review we focus on two major anti-apoptotic protein families: IAPs (Inhibitor of Apoptosis) proteins and Bcl-2 (B-cell lymphoma-2) family members. We also discuss insights into the regulation of these proteins by natural antagonists, which has provided the conceptual basis for developing novel anti-cancer drugs. Significantly, the pro-apoptotic protein ARTS (apoptosis-related protein in the TGF-β signaling pathway; Sept4_i2) acts as a dual antagonist of both X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2. Because upregulation of anti-apoptotic proteins in response to cancer therapy contributes to drug resistance, targeted inhibition of these proteins is expected to enhance the efficacy of chemotherapy. Finally, we discuss the role of proteasome-mediated degradation in the regulation of apoptosis, and how this mechanism can be harnessed to develop small molecules that stimulate degradation of anti-apoptotic proteins for cancer therapy. This strategy has the potential to overcome drug resistance more effectively than mere inhibition. Therefore, this approach may allow use of lower drug concentrations and thereby reduce cytotoxicity and untoward side effects.

In Vitro and In Vivo Antioxidant Activities of Polysaccharides Isolated from Celluclast-Assisted Extract of an Edible Brown Seaweed, Sargassum fulvellum.

It has been reported that enzymatic digestion of algae could improve the yield and enhance the biological activity compared to water and organic extraction. Our previous research indicated that Celluclast-assisted extract of Sargassum fulvellum (SF) possessed higher carbohydrate content and stronger antioxidant activity compared to water and other enzyme-assisted extracts. In the present study, we evaluated the antioxidant activities of polysaccharides from SF (SFPS) in vitro in Vero cells and in vivo in zebrafish. SFPS was obtained by Celluclast-assisted hydrolysis and ethanol precipitation. Results showed that SFPS contained 74.55 ± 1.26% sulfated polysaccharides and effectively scavenged 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and alkyl radicals. SFPS significantly and dose-dependently scavenged intracellular reactive oxygen species (ROS) and improved cell viability. Further studies indicated that SFPS reduced apoptotic body formation through downregulation of proapoptotic protein (Bax and cleaved caspase-3) levels and upregulation of antiapoptotic protein (Bcl-xL and PARP) levels in 2,2-azobis(2-amidinopropane) hydrochloride (AAPH)-treated Vero cells. In addition, SFPS showed strong protective effect against AAPH-stimulated oxidative stress in vivo in zebrafish, as demonstrated by the improved survival rate, reduced heart rate, and decrease in ROS, cell death, and lipid peroxidation levels. These results suggest that SFPS possesses strong in vitro and in vivo antioxidant activity and can be a potential ingredient in the pharmaceutical and cosmeceutical industries.

Blocking lncRNA MALAT1/miR-199a/ZHX1 Axis Inhibits Glioblastoma Proliferation and Progression

Zinc fingers and homeoboxes 1 (ZHX1) is a transcription repressor that has been implicated in the tumorigenesis and progression of diverse tumors. The functional role and regulating mechanism of ZHX1 has not been elucidated in glioblastoma (GBM). Previous reports have suggested that a large number of non-coding RNAs play a vital role in glioma initiation and progression. This study aimed to investigate the functional role and co-regulatory mechanisms of the metastasis-associated lung adenocarcinoma transcript-1 (MALAT1)/ microRNA-199a (miR-199a)/ZHX1 axis in GBM. We analyzed the expression of the MALAT1/miR-199a/ZHX1 axis and its correlation with patients’ overall survival using two different glioma gene-expression datasets. A series of in vitro and in vivo studies including dual luciferase reporter assay, fluorescence in situ hybridization (FISH), RNA immunoprecipitation, and pull-down experiments were completed to elucidate the biological significance of the MALAT1/miR-199a/ZHX1 axis in promoting glioma proliferation and progression. Elevated ZHX1 expression correlated with poor prognosis in GBM patients, and in vitro studies demonstrated that ZHX1 attenuated GBM cell apoptosis by downregulation of pro-apoptotic protein (Bax) and upregulation of anti-apoptotic protein (Bcl-2). Furthermore, knockdown of MALAT1 inhibited GBM proliferation and progression in vitro and reduced tumor volume and prolonged survival in an orthotopic GBM murine model. Finally, we demonstrated that MALAT1 promoted ZHX1 expression via acting as a competing endogenous RNA by sponging miR-199a. The MALAT1/miR-199a/ZHX1 axis promotes GBM cell proliferation and progression in vitro and in vivo, and its expression negatively correlates with GBM patient survival. Blocking the MALAT1/miR-199a/ZHX1 axis can serve as a novel therapeutic strategy for treating GBM.