OM301, a Synthetic Polypeptide Containing the p53TA (Transactivation) Domain, Impairs Mitochondrial Activity and Survival of Myeloma Cells

Abstract INTRODUCTION: Although the treatment of patients with multiple myeloma (MM) has dramatically improved, those with high-risk characteristics, including the deletion or mutation of the master tumor suppressor gene TP53 on chromosome 17, experience limited survival. OM301 is a synthetic polypeptide containing the p53TA (transactivation) domain, which prevents p53 degradation through inhibition of MDM2. Here, we demonstrate that OM301 has strong anti-MM activity in vitro and in vivo. RESULTS: We first assessed the cytotoxic effects of OM301 in MM cell lines with varying TP53 status (TP53 wild type: MM.1S, H929; TP53 mutated/null: L363, RPMI-8226, U266, JJN3, KMS11) and found that OM301 exerts significant cytotoxic effects at a concentration of ~5 µM in all cell lines we tested, while it was minimally toxic to human peripheral blood mononuclear cells. Next, using immunocompromised NSG mice models injected with MM.1S, we determined the in vivo efficacy of OM301 in three different studies. Many potent anticancer agents, particularly of peptide origin, show prominent anti-tumor effects but fail to sustain similar effects when given intraperitoneally because of poor absorption, distribution, metabolism and excretion properties. OM301 at an intraperitoneal dose of 20 mg/kg/body weight twice a day induced significant reduction in tumor size with respect to vehicle control, suggesting the stability of OM301 without any loss of its activity (n=7, p<0.0001). Accordingly, we investigated its effect in a disseminated NSG/MM.1S model and found that it significantly increased survival (p<0.0001) (see Figure). Because OM301 was designed to simulate the p53 interaction domain with MDM2, we first determined its effect on p53-MDM2 crosstalk using a p53-MDM2 co-Immunoprecipitation (co-IP) assay and compared it with effects from Nutlin-3a, a known inhibitor of p53-MDM2. The co-IP data showed that, unlike Nutlin-3a, OM301 does not inhibit the p53-MDM2 interaction. Thus, to confirm our findings, we first overexpressed MDM2 in HeLa cells, and, using MDM2-IP and p53-MDM2 co-IP, found similar observations. Additionally, OM301 also failed to induce endogenous upregulation of genes activated by p53, such as MDM2 and p21, as opposed to results from Nutlin-3a. RNA sequencing data also showed a distinctive OM301signature, as compared to Nutlin-3a in MM cells. While treatment of Nutlin-3a induced expression of p53-activated canonical genes, OM301-treated cells showed alterations in genes involved in inflammatory responses, c-Myc regulated genes, fatty acid metabolism, glucose metabolism, and oxidative phosphorylation, among others. Next, to dissect its underlying mechanism, we dual-tagged OM301 with fluorophores at the 3' and 5' ends to study its localization and its stability in MM cells. Indeed, OM301 was found to be stable and mainly localized in the cytosol. We then modified OM301 by biotinylation of its penetratin end and first verified its cytotoxic effect in different MM cell lines, which was similar to that of native OM301. The biotinylated OM301 was then immunoprecipitated using streptavidin beads. The streptavidin pull-down and subsequent proteomic analysis confirmed that OM301 does not interact with MDM2 but interacts with c-Myc and with proteins localized in mitochondria, including Bcl-2 and Bcl-2 family members such as Bclaf1, Bcl2L13, and Bcl2L1. Pull-down experiments and immunoblot analysis validated Bcl-2/OM301 interactions. To further evaluate the relative binding potentials of OM301, we performed molecular docking studies using the HPEPDOCK server (Yan et al., Nat Protoc. 2020;15:1829). Post-docking, the calculated docking scores for OM301 was -281, suggesting that OM301directly interacts with Bcl-2. Thus, we evaluated the effects of OM301 on mitochondrial function and physiology. Treatment with OM301 decreased mitochondrial membrane potential in different MM cell lines. OM301 also increased mitochondrial superoxide production and induced mitophagy and mitochondrial fission as seen by electron microscopy. CONCLUSION: Here, we report for the first time that OM301, although designed for p53-selective cells, may instead interact with Bcl-2, which in turn induces mitochondrial dysfunction, leading to cell death irrespective of their TP53 status. Our data suggest that OM301 may be a novel and effective therapeutic option for MM. Figure 1 Figure 1. Disclosures Krishnan: REGENERON: Consultancy; MAGENTA: Consultancy; BMS: Consultancy, Current equity holder in publicly-traded company, Speakers Bureau; JANSSEN: Consultancy, Research Funding; City of Hope Cancer Center: Current Employment; SANOFI: Consultancy; GSK: Consultancy; Amgen: Speakers Bureau. Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

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Cytotoxic Effects of 24/R,25-Dihydroxycholecalciferol on the Proliferation of Various Tumor Cell Lines in vitro and Its Antitumor Effects in vivo

Oncology ◽

10.1159/000226563 ◽

1988 ◽

Vol 45 (3) ◽

pp. 206-209 ◽

Cited By ~ 1

Author(s):

Yuji Maeda ◽

Tohru Hirai ◽

Hideyuki Yamato ◽

Noriko Kobori ◽

Ken-ichi Matsunaga ◽

...

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Tumor Cell Lines ◽

Antitumor Effects ◽

Cytotoxic Effects

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Identification of Tight Junction Protein (TJP)-1 As a Modulator of Sensitivity to Doxorubicin and Cisplatin in Models of Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.3962.3962 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3962-3962

Author(s):

Xing-Ding Zhang ◽

Robert Z. Orlowski ◽

Lin Yang

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Hela Cells ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Cancer Center ◽

In Vivo Models ◽

Junction Protein ◽

Rpmi 8226

Abstract Abstract 3962 Background: Therapeutic advances in multiple myeloma have improved the outcomes of patients with this malignant plasma cell disorder, but the disease course is still strongly influenced by both innate, or primary, as well as acquired, or secondary mechanisms of drug resistance. Identification and validation of genes that may mediate these phenotypes is therefore of importance, since they could be useful prognostic markers, and also potential targets to overcome the emergence of resistance, or possibly preclude its emergence altogether. Methods: To identify non-redundant determinants of chemoresistance, we designed a robust, high-throughput RNA interference (RNAi) screen targeting 9610 human genes. The screen involved retroviral-mediated transduction first of HeLa cervical carcinoma cells with either the RNAi library, or with non-targeting retrovirus particles. After infection, cells were selected with puromycin, and treated with different concentrations of doxorubicin and cisplatin. Doxorubicin (Dox) treatment led to 33 surviving colonies from the cells transduced with the shRNA library, cisplatin (Cis) treatment led produced 22 surviving colonies, while non-targeting retrovirus-infected cells failed to form colonies after treatment. Screening was performed to identify the shRNA target gene(s) in each colony, and genes that were identified in both Dox- and Cis-treated HeLa cells, and that were expressed in myeloma cells, were selected for further study. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results: TJP1 (zona occludens (ZO)-1) was identified as one gene whose knockdown promoted survival in Dox- and Cis-treated HeLa cells, and which was expressed in myeloma cell lines and in primary plasma cells. To further examine its potential role in myeloma chemosensitivity, we performed mRNA and protein expression profiling in a panel of 11 cell lines and observed that TJP1 expression was silenced in 3 cell lines (ARP-1, INA-6, and MOLP-8), while it was moderately to highly expressed in 7 cell lines (including RPMI 8226, MM1.S, and U266). Comparing TJP1-positive MM1.S cells to TJP1-null MOLP-8 cells, the latter displayed a significantly higher median inhibitory concentration to Dox and Cis. Knockdown of TJP1 in RPMI 8226 and U266 cells, which produced a >75% target suppression, was sufficient to reduce the proportion of apoptotic cells in the sub-G1 fraction after treatment with Dox or Cis compared to control cells. Conversely, MOLP-8 cells transfected with human TJP1 cDNA exhibited an increase in the sub-G1 population in response to Dox and Cis treatment compared to vector controls. Conclusion: Taken together, these studies support the hypothesis that TJP1 expression mediates myeloma cell resistance to the DNA damaging agents doxorubicin and cisplatin. Further studies are underway to determine the mechanism by which TJP1 influences chemosensitivity, and to validate its impact using in vivo models. Disclosures: No relevant conflicts of interest to declare.

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Anti-Myeloma Activity by the Combination of the JAK2 Inhibitor Ruxolitinib with Lenalidomide and Corticosteroids

Blood ◽

10.1182/blood.v124.21.2114.2114 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2114-2114 ◽

Cited By ~ 2

Author(s):

Haiming Chen ◽

Eric Sanchez ◽

Mingjie Li ◽

Cathy Wang ◽

Abby Gillespie ◽

...

Keyword(s):

Tumor Growth ◽

Cell Viability ◽

Tumor Cells ◽

Cell Lines ◽

Scid Mice ◽

M Protein ◽

Cytotoxic Effects ◽

Jak2 Inhibitor

Abstract Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV). Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs. Results: In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone. Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

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Mechanism of cellular production and in vivo seeding effects of hexameric β-amyloid assemblies

10.1101/2020.12.23.424094 ◽

2020 ◽

Author(s):

Céline Vrancx ◽

Devkee M Vadukul ◽

Sabrina Contino ◽

Nuria Suelves ◽

Ludovic D’Auria ◽

...

Keyword(s):

Cell Lines ◽

Amyloid Deposition ◽

Amyloid Peptide ◽

Intracerebral Injection ◽

Cytotoxic Effects ◽

Wide Range ◽

Β Amyloid ◽

5Xfad Mice

AbstractBackgroundThe β-amyloid peptide (Aβ) plays a key role in Alzheimer’s disease. After its production by catabolism of the amyloid precursor protein (APP) through the action of presenilin 1 (PS1)- or presenilin 2 (PS2)-dependent γ-secretases, monomeric Aβ can assemble in oligomers. In a pathological context, this eventually leads to the formation of fibrils, which deposit in senile plaques. Many studies suggest that Aβ toxicity is related to its soluble oligomeric intermediates. Among these, our interest focuses on hexameric Aβ, which acts as a nucleus for Aβ self-assembly.MethodsBiochemical analyses were used to identify hexameric Aβ in a wide range of models; cell lines, cerebrospinal fluid from cognitively impaired patients and transgenic mice exhibiting human Aβ pathology (5xFAD). We isolated this assembly and assessed both its effect on primary neuron viability in vitro, and its contribution to amyloid deposition in vivo following intracerebral injection. In both cases, we used wild-type mice (C57BL/6) to mimic an environment where hexameric Aβ is present alone and 5xFAD mice to incubate hexameric Aβ in a context where human Aβ species are pre-existing. Using CRISPR-Cas9, we produced stable knockdown human cell lines for either PS1 or PS2 to elucidate their contribution to the formation of hexameric Aβ.ResultsIn WT mice, we found that neither in vitro or in vivo exposure to hexameric Aβ was sufficient to induce cytotoxic effects or amyloid deposition. In 5xFAD mice, we observed a significant increase in neuronal death in vitro following exposure to 5μM hexameric Aβ, as well as a 1.47-fold aggravation of amyloid deposition in vivo. At the cellular level, we found hexameric Aβ in extracellular vesicles and observed a strong decrease in its excretion when PS2 was knocked down by 60%.ConclusionsOur results indicate the absence of cytotoxic effects of cell-derived hexameric Aβ by itself, but its capacity to aggravate amyloid deposition by seeding other Aβ species. We propose an important role for PS2 in the formation of this particular assembly in vesicular entities, in line with previous reports linking the restricted location of PS2 in acidic compartments to the production of more aggregation-prone Aβ.

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The Effects of Combining Cancer Drugs with Compounds Isolated from Combretum zeyheri Sond. and Combretum platypetalum Welw. ex M.A. Lawson (Combretaceae) on the Viability of Jurkat T Cells and HL-60 Cells

BioMed Research International ◽

10.1155/2021/6049728 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Morris Wende ◽

Simbarashe Sithole ◽

Godloves Fru Chi ◽

Marc Y. Stevens ◽

Stanley Mukanganyama

Keyword(s):

T Cells ◽

Cell Lines ◽

Cancer Cell ◽

Mammalian Cells ◽

Cancer Cell Lines ◽

Jurkat T Cells ◽

Cytotoxic Effects ◽

Peritoneal Cells

Combretum zeyheri and Combretum platypetalum have been shown to have anticancer, antibacterial, antituberculosis, and antifungal effects in both in vivo and in vitro studies. This study sought to evaluate the antiproliferative effects of compounds isolated from C. zeyheri and C. platypetalum on Jurkat T and HL-60 cancer cell lines in combination with doxorubicin and/or chlorambucil. At their GI50 concentrations, the isolated compounds were combined with the corresponding GI50 of chlorambucil and doxorubicin. The cytotoxic effects of the combined compounds were determined on BALB/c mouse peritoneal cells. All the 4 isolated compounds had significant cytotoxic effects on Jurkat T cells. Compounds CP 404 (1), CP 409 (2), CZ 453 (3), and CZ 455 (4) had GI50s on Jurkat T cells of 3.98, 19.33, 6.82, and 20.28 μg/ml, respectively. CP 404 (1), CP 409 (2), CZ 453 (3), and CZ 455 (4) showed GI50s of 14.18, 28.69, 29.87, and 16.46 μg/ml on HL-60 cancer cell lines, respectively. The most potent combination against Jurkat T cells was found to be CP 404 (1) and chlorambucil. This combination showed no cytotoxic effects when tested on BALB/c mouse peritoneal cells. It was concluded that the compounds extracted from C. zeyheri and C. platypetalum inhibit the growth of Jurkat T cells in vitro. The combination of the compounds with anticancer drugs enhanced their anticancer effects. The combination of CP 404 (1) and chlorambucil was found not to be toxic to normal mammalian cells. Therefore, CP 404 (1), 3-O-β-L-rrhamnopyranosyl-5,7,3 ′ 4 ′ ,5 ′ -pentahydroxyflavone, has the potential to be a source of lead compounds that can be developed for anticancer therapy. Further structure-activity relationship studies on this compound are warranted.

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In Vitro Assessment of Anti-Multiple Myeloma (MM)-Agents On Various MM-Cell Lines (MMCLs) with Use of Bortezomib, Sorafenib, Thalidomide, Lenalidomide and EpiGalloCatechin-3-Gallate (EGCG) Demonstrates to Be a Valuable Tool for the Thorough Analysis and Discovery of Innovative Anti-MM-Agents.

Blood ◽

10.1182/blood.v114.22.4922.4922 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4922-4922

Author(s):

Josefina Udi ◽

Dagmar Wider ◽

Gabriele Ihorst ◽

Julia Schüler ◽

Ralph Wäsch ◽

...

Keyword(s):

Cell Growth ◽

Confocal Microscopy ◽

Cell Viability ◽

Cell Lines ◽

Cytotoxic Effects ◽

Inhibition Of Proliferation ◽

Viable Cells ◽

Morphologic Changes

Abstract Abstract 4922 Introduction Thalidomide and lenalidomide possess antiangiogenic, antiproliferative, proapoptotic and immunomodulatory effects. The proteasome inhibitor bortezomib induces cell death in MMCLs and has demonstrated synergism on various tumor cell lines, when combined with the multikinase inhibitor sorafenib. Sorafenib targets various kinases involved in tumor growth and angiogenesis, which plays a governing role in numerous cancers, including MM. EGCG, the most active catechin in green tea, has been described to induce anti-MM- and anti-amyloid-, but recently also to prevent bortezomib-induced-effects. We therefore tested these compounds individually and in combinations on 3 MMCLs in order to assess their cytotoxicity, cell growth inhibition and phenotype changes. Material and Methods RPMI8226, U266 and L363 were cultured at 1×105 cells/ml, with RPMI1640, 10% FCS and 0.2% pen/strep. On day (d) 0, increasing concentrations of bortezomib, sorafenib, thalidomide, lenalidomide and EGCG were added. On d3 and d6, we determined cytotoxicity and cell viability via trypan blue dye exclusion assay and propidium iodide (PI) staining by flow-cytometry (FACS). Additionally, we analyzed phenotype changes by means of CD138-expression (FACS). To evaluate CD138-expression as well as morphologic changes after sorafenib treatment, we also performed confocal microscopy analyses. Results 100nM bortezomib showed pronounced cytotoxicity on all 3 MMCLs: mean PI-positivity in L363 was 83.9% on d3, remained stable on d6 and was significantly increased as compared to control-L363-cells (p<0.01). In U266, mean PI-positivity on d3 and d6 was 37.5% and 25.2%, respectively, again being significantly higher than in control-U266-cells (p<0.0001). In RPMI, PI-positivity was similarly increased on d3 and d6 with 94.5% and 91.3%, respectively, again substantially higher than in control-RPMI-cells (p<0.001). With 10 and 100μM sorafenib in L363, we observed mean PI+ cells on d3 as high as 61.6% and 94.3% and on d6 of 80.8% and 91.8%, respectively (p<0.0001). A statistically significant dose-dependent decrease in viable cells and CD138-expression with 10 and 100μM sorafenib as compared to L363-control-cells could also be detected. By confocal microscopy, CD138-downregulation was prominent, besides manifest morphologic changes. In U266, mean PI+ cells were 33.1% with 10μM and 78.3% with 100μM, again significantly higher than in control-U266-cells (p<0.001), not substantially increasing on d6. Sorafenib's cytotoxicity was likewise evident in RPMI: PI+ cells on d3 with 10 and 100μM were 89.8% and 95.2% (p<0.001), respectively. In contrast, even with 100μg/ml thalidomide, cytotoxicity in L363 cells was subtle, with mean PI+ cells of 10.0% on d3 and 21.6% on d6 (n.s.), with cell viability only slightly decreasing. Thalidomide did not significantly affect U266 cell growth either. Lenalidomide did not increase PI+ cells in L363 on d3, but induced a noticeable PI+-rise on d6 of 19.2% with 10μM (p<0.01) and 15% with 100μM (p<0.001) as compared to control-L363-cells. No statistically significant decrease in viable cells and CD138-expression was observed. With 10 and 100μM lenalidomide in U266, PI+ cells on d6 were higher with 19.9% and 29.6% (p<0.001), respectively, as compared to control-U266-cells. Exposure of all MMCLs to EGCG, with concentrations ranging from 1 to 500μM, showed potent cytotoxic effects, most evident with concentrations of 250μM or higher in L363 (p<0.01). Conclusions Bortezomib and sorafenib showed impressive cytotoxic effects as single agents and ongoing experiments suggest additive effects between both compounds, which is currently being investigated, both in vitro and in our in vivo NOD/SCID-IL2-receptor-gamma-chain−/− (NSG)-mouse-model. Further investigations will also validate the recently suggested inhibitory effects of EGCG on bortezomib-induced cytotoxicity. Thalidomide and lenalidomide moderately reduced viable cell numbers, confirming that other mechanisms, such as anti-angiogenesis and immunomodulation are of greater relevance on MM cells. In line with earlier work, EGCG induced a pronounced cytotoxic effect and inhibition of proliferation. Our results demonstrate that our in vivo model is valuable for the thorough analysis and discovery of innovative targeted anti-MM-agents. Disclosures No relevant conflicts of interest to declare.

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Potential Use of Sphingosine Kinase-2 Selective Inhibitors for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.5105.5105 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5105-5105

Author(s):

Ningfei An ◽

Yeong-Bin Im ◽

Peng Gao ◽

Luciano J Costa ◽

Robert K Stuart ◽

...

Keyword(s):

Pancreatic Cancer ◽

Multiple Myeloma ◽

Cell Proliferation ◽

Cell Lines ◽

Therapeutic Potential ◽

Myeloma Cell ◽

Cancer Center ◽

Selective Inhibitors ◽

Myeloma Cells

Abstract Abstract 5105 Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. Despite the widespread use of several highly active chemotherapy agent (thalidomide, lenalidomide and bortezomib) and the incorporation of autologous hematopoietic stem cell transplantation, MM remains an incurable disease, suggesting the need for a better understanding of the disease's molecular pathways and for the development of novel agents. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. Among a series of sphingolipid-metabolizing enzymes, sphingosine kinases (SK-1 and -2) are considered to be key regulators of cancer cell proliferation. SK phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and affects the delicate balance between apoptotic ceramide/sphingosine and proliferative S1P. SKs are highly expressed in several solid tumors including pancreatic cancer and ovarian cancer. Currently, however, very little is known about how the SK is expressed in MM, and how sphingolipids respond to drugs targeting SK. We hypothesize that SKs play important roles in the pathogenesis of MM, thus providing a novel target for the treatment of MM. Apogee Biotechnology Corporation has generated new, orally bioavailable small molecule SK inhibitors that have in vitro and in vivo activity in a variety of models of cancer and inflammatory diseases. Several advantages of these SK inhibitors over other anti-MM drugs include: the role of the target in multiple key pathways driving MM; the low toxicity observed in GLP toxicology testing; the oral bioavailability that will simplify administration; and the ability to be combined with the standard drugs for MM (i.e, bortezomib). ABC294640, a SK2-selective inhibitor has recently entered single-agent clinical testing in advanced pancreatic cancer at our institution. In the current study, we investigated the expression patterns of SK2 in myeloma cells and the therapeutic potential of ABC294640 in the treatment of MM. We found that SK2 is highly expressed in several myeloma cell lines and in primary human CD138+ myeloma cells. Compared to the A498 human kidney adenocarcinoma cell line, the expression levels of SK2 mRNA were 1.4–12 fold higher in myeloma cell lines. The expression level of SK2 mRNA was > 62 fold higher in primary human CD138+ myeloma cells, compared to CD138- cells (n = 4). When myeloma cells were treated with ABC294640, cell proliferation was effectively inhibited with IC50 of ∼20 μM, including steroid resistant MM1.R myeloma cells. The degree of cell growth inhibition by ABC294640 correlated well with the expression level of SK2 mRNA in the myeloma cells. We also found that ABC294640 induces PARP cleavage and caspase 3 and 9 activation, indicating that the SK2 inhibitor induces apoptotic cell death. We are currently testing the effects of ABC294640 alone and in combination with dexamethasone or proteasome inhibitors in vivo in the XBP-1s transgenic MM model. Preclinical toxicities of the drug combinations will also be determined in the mouse models. Our studies provide the first evidence of SK2 in the pathogenesis of MM, and suggest excellent therapeutic potential of SK2-selective inhibitors for the treatment of MM. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.

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CD95L Inhibition Impacts Gemcitabine-Mediated Effects and Non-Apoptotic Signaling of TNF-α and TRAIL in Pancreatic Tumor Cells

Cancers ◽

10.3390/cancers13215458 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5458

Author(s):

Khalid Rashid ◽

Christian Röder ◽

Freya Goumas ◽

Jan-Hendrik Egberts ◽

Holger Kalthoff

Keyword(s):

Tumor Cells ◽

Cell Lines ◽

Inflammatory Responses ◽

Inhibitory Effect ◽

Cell Phenotype ◽

Mediated Effects ◽

Tnf Α ◽

Trail Signaling ◽

Specific Impact

Despite the potential apoptotic functions, the CD95/CD95L system can stimulate survival as well as pro-inflammatory signaling, particularly through the activation of NFκB. This holds true for the TNF/TNFR and the TRAIL/TRAILR systems. Thus, signaling pathways of these three death ligands converge, yet the specific impact of the CD95/CD95L system in this crosstalk has not been well studied. In this study, we show that gemcitabine stimulates the expression of pro-inflammatory cytokines, such as IL6 and IL8, under the influence of the CD95/CD95L system and the pharmacological inhibitor, sCD95Fc, substantially reduced the expression in two PDAC cell lines, PancTuI-luc and A818-4. The stem cell phenotype was reduced when induced upon gemcitabine as well by sCD95Fc. Moreover, TNF-α as well as TRAIL up-regulate the expression of CD95 and CD95L in both cell lines. Conversely, we detected a significant inhibitory effect of sCD95Fc on the expression of both IL8 and IL6 induced upon TNF-α and TRAIL stimulation. In vivo, CD95L inhibition reduced xeno-transplanted recurrent PDAC growth. Thus, our findings indicate that inhibition of CD95 signaling altered the chemotherapeutic effects of gemcitabine, not only by suppressing the pro-inflammatory responses that arose from the CD95L-positive tumor cells but also from the TNF-α and TRAIL signaling in a bi-lateral crosstalk manner.

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Abemaciclib in Combination with Copanlisib to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-150942 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2253-2253

Author(s):

Yuxuan Che ◽

Yang Liu ◽

Yijing Li ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

...

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Clinical Care ◽

Single Agent ◽

Xenograft Model ◽

Cancer Center ◽

Cell Viability Assay

Abstract Introduction Although novel therapeutic strategies including BTK and Bcl-2 inhibitors have dramatically improved the prognosis of MCL patients, resistance to these treatments is inevitable. We recently reported that the tumor suppressor gene CDKN2A were commonly deleted in ibrutinib-resistant tumors, leading to upregulation of CDK4/6 signaling. Among the other hallmarks are the mTOR/PI3K, Myc and OXPHOS pathways. Therefore, we attempt to exploit combinatory targeting of CDK4/6 and PI3K pathways to overcome therapy resistance using in vitro and PDX models. Methods Ibrutinib or venetoclax sensitive and resistant MCL cell lines were used in this preclinical study. 1x10 4 cells per well are seeded in 96-well plates and treated with abemaciclib monotherapy or in combination with copanlisib (PI3K inhibitor) in triplicate for 72h and then mixed with CellTiter-Glo Luminescent Cell viability Assay Reagent. For cell cycle assay, cells were seeded in 6 well plates and treated with vehicle or abemaciclib for 24h. Cells were fixed in 70% pre-cold ethanol and stained with propidium iodide. The cell cycle stages were quantified through the Novocyte Flow Cytometer. The molecular events at the protein level after treatment were determined by immunoblotting. For in vivo experiment, the combination of abemaciclib (25mg/kg, oral, daily) and copanlisib (5mg/kg, IP, three times a week) was assessed in Mino-venetoclax-resistant xenograft model. IC50 values were calculated using GraphPad Prism 8 for each cell line. Student's t-test was performed to compare the difference between vehicle and treated groups. Two-way analysis of variance (ANOVA) was conducted to analyze the tumor growth in vivo experiments. P values less than 0.05 were considered statistically significant. Results Our previous studies have identified a subset of MCL cells that were resistant to venetoclax (JeKo-1) or ibrutinib treatment (Maver-1 and Z-138). To overcome the resistance, we first treated MCL cell lines with abemaciclib and the result showed that abemaciclib as a single agent showed potent anti-MCL activity in a subset of MCL cell lines (IC 50 = 70-952 nM) including venetoclax sensitive- (Mino, Rec-1, Maver-1, and Z138) and primary resistant- MCL cells (JeKo-1). However, the cell lines Mino-ven-R and Rec-ven-R with acquired venetoclax resistance are highly resistant to abemaciclib treatment (IC 50 = 6.0 and 4.4 µM). PI3K/ATK pathway has been reported to be highly upregulated in Mino-ven-R and Rec-ven-R cells compared to their parental cells. To further increase the efficacy of the targeted therapy, we treated the resistant MCL cells with a combination of abemaciclib and copanlisib and the result showed synergistically enhanced cytotoxicity in ibrutinib or venetoclax-resistant MCL cell lines. Consistent with the role of CDK4/6 in cell cycle progression, inhibition of CDK4/6 with abemaciclib resulted in the cell cycle arrest at G1 phase in MCL cell lines. To validate whether abemaciclib in combination with copanlisib can overcome venetoclax resistance in vivo, we assessed the antitumor effect of abemaciclib in combination with copanlisib using a venetoclax-resistant xenograft models derived from Mino-ven-R cell line in immunodeficient NSG mice. As a result, abemaciclib (25 mg/kg, oral, daily), but not venetoclax (5 mg/kg, oral, daily) or copanlisib (5 mg/kg, IP, three times a week), significantly reduced tumor volume compared to the vehicle control (n = 5, p < 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p<0.0001). Of note, the combination did not cause major decreases in body weight. Taken together, these results suggest that the combinatory therapy is effective in overcoming venetoclax resistance in MCL. Conclusions Combinatory treatment with abemaciclib and copanlisib may achieve clinical actionable efficacy through overcoming the venetoclax-resistance in MCL that may become an effective treatment regimen for refractory/relapsed MCL patients in the future. Disclosures Wang: Dava Oncology: Honoraria; Imedex: Honoraria; CStone: Consultancy; Hebei Cancer Prevention Federation: Honoraria; OMI: Honoraria; Chinese Medical Association: Honoraria; Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; Bayer Healthcare: Consultancy; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; Physicians Education Resources (PER): Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; Acerta Pharma: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; BGICS: Honoraria; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding.

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Mechanism of Cellular Production and in Vivo Seeding Effects of Hexameric β-Amyloid Assemblies

10.21203/rs.3.rs-138906/v1 ◽

2021 ◽

Author(s):

Céline Vrancx ◽

Devkee M Vadukul ◽

Sabrina Contino ◽

Nuria Suelves ◽

Ludovic D'Auria ◽

...

Keyword(s):

Cell Lines ◽

Amyloid Deposition ◽

Amyloid Peptide ◽

Intracerebral Injection ◽

Cytotoxic Effects ◽

Wide Range ◽

Β Amyloid ◽

5Xfad Mice

Abstract BackgroundThe β-amyloid peptide (Aβ) plays a key role in Alzheimer’s disease. After its production by catabolism of the amyloid precursor protein (APP) through the action of presenilin 1 (PS1)- or presenilin 2 (PS2)-dependent γ-secretases, monomeric Aβ can assemble in oligomers. In a pathological context, this eventually leads to the formation of fibrils, which deposit in senile plaques. Many studies suggest that Aβ toxicity is related to its soluble oligomeric intermediates. Among these, our interest focuses on hexameric Aβ, which acts as a nucleus for Aβ self-assembly.MethodsBiochemical analyses were used to identify hexameric Aβ in a wide range of models; cell lines, cerebrospinal fluid from cognitively impaired patients and transgenic mice exhibiting human Aβ pathology (5xFAD). We isolated this assembly and assessed both its effect on primary neuron viability in vitro, and its contribution to amyloid deposition in vivo following intracerebral injection. In both cases, we used wild-type mice (C57BL/6) to mimic an environment where hexameric Aβ is present alone and 5xFAD mice to incubate hexameric Aβ in a context where human Aβ species are pre-existing. Using CRISPR-Cas9, we produced stable knockdown human cell lines for either PS1 or PS2 to elucidate their contribution to the formation of hexameric Aβ.ResultsIn WT mice, we found that neither in vitro or in vivo exposure to hexameric Aβ was sufficient to induce cytotoxic effects or amyloid deposition. In 5xFAD mice, we observed a significant increase in neuronal death in vitro following exposure to 5μM hexameric Aβ, as well as a 1.47-fold aggravation of amyloid deposition in vivo. At the cellular level, we found hexameric Aβ in extracellular vesicles and observed a strong decrease in its excretion when PS2 was knocked down by 60%.ConclusionsOur results indicate the absence of cytotoxic effects of cell-derived hexameric Aβ by itself, but its capacity to aggravate amyloid deposition by seeding other Aβ species. We propose an important role for PS2 in the formation of this particular assembly in vesicular entities, in line with previous reports linking the restricted location of PS2 in acidic compartments to the production of more aggregation-prone Aβ.

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