Pre-Clinical Evaluation of the PI3K-p110β/δ Inhibitor KA2237 in Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1837-1837 ◽  
Author(s):  
Shengjian Huang ◽  
Loretta J. Nastoupil ◽  
Hui Guo ◽  
Taylor Bell ◽  
Makhdum Ahmed ◽  
...  
Keyword(s):  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Synergistic Effects ◽  
Resistant Cell ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Mantle Cell ◽  
Dose Dependent

Abstract Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma and is a therapeutic challenge. Phosphoinositide-3 kinase (PI3K) has been shown to be an alternative survival pathway in relapsed/refractory MCL. KA2237 (designed by Karus Therapeutics Ltd, Oxfordshire, United Kingdom) is a dual inhibitor of the class I beta and delta isoforms of the 110 kDa catalytic subunit of PI3K. By selectively targeting PI3K-beta and -delta isoforms and preventing their activation, KA2237 may decrease proliferation and induce cell death in susceptible tumor cells. Methods: We assessed the effects of KA2237 on the in vitro cell proliferation of both ibrutinib-sensitive (Mino, Jeko-1, and Rec-1) and primary ibrutinib-resistant (Z-138 and Maver-1) cell lines, and acquired ibrutinib-resistant MCL cell line, Jeko-R. We also tested the viability of patient-derived xenograft (PDX) tumor cells to KA2237. We compared the efficacy of KA2237 with two other commercial PI3K inhibitors, duvelisib (IPI-145, Selleck) and idelalisib (Cal-101, Selleck). Also, we paired these three inhibitors (KA2237, duvelisib and idelalisib) each with ibrutinib to evaluate the potential synergistic effects of these combinations. Lastly, we also tested in vivo efficacy of KA2237 and its combination with ibrutinib in PDX tumor cells. Results: KA2237 inhibited cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent and time-dependent manner. For Mino and Jeko-1, the IC50 was 4.8 uM and 2.9 uM and for Z-138 and Maver-1 cell lines, the IC50 was 0.6 uM and 0.1 uM, respectively. KA2237 also decreased cell viability of ibrutinib-sensitive and ibrutinib-resistant MCL PDX tumor cells. However, KA2237 did not decrease the cell viability of normal human peripheral blood mono-nuclear cells. KA2237 arrested phase G0/G1 in Rec-1 and Jeko-R cell lines. We detected the expression of PI3K isoforms in MCL, finding higher expression of PI3K β and δ in MCL-resistant cell lines as compared with sensitive cell lines. We found that KA2237 induced MCL cell apoptosis in a time-dependent and dose-dependent manner. In comparison with duvelisib and idelalisib, KA2237 achieved greater inhibition of cell viability, cell apoptosis and cell cycle arrest. Furthermore, we found synergistic effects of KA2237 and ibrutinib combination in several MCL cell lines and in PDX models. In an ibrutinib-resistant PDX model, KA2237 treated mice reduced tumor burden significantly compared with vehicle control, and higher tumor growth inhibition was achieved as compared with ibrutinib. Conclusion: The novel PI3K inhibitor, KA2237 may be a potential candidate for MCL therapy, especially in the ibrutinib-resistant cases. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding; BeiGene: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Celgene: Research Funding.

scholarly journals Inhibition of B-Cell Receptor Signaling Disrupts Cell Adhesion in Mantle Cell Lymphoma Via RAC2

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1369-1369
Author(s):  
Weige Wang ◽  
Franzen Carrie ◽  
Hui Guo ◽  
Jimmy Lee ◽  
Yan Li ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
Resistant Cell ◽  
Advisory Committees ◽  
Tumor Tissues ◽  
Mantle Cell ◽  
Bcr Signaling

Abstract Background: B-cell receptor (BCR) signaling pathway is recognized as a crucial pathway for the pathogenesis of neoplastic B-cells. Inhibition of the BCR signaling and the downstream pathway is highly effective in B-cell malignancy through Bruton tyrosine kinase inhibition by ibrutinib. In addition to cell proliferation inhibition, ibrutinib disrupts cell adhesion between tumor and its microenvironment through unknown molecular mechanisms, resulting in peripheral lymphocytosis with accompanying lymphadenopathy reduction in patients who receive ibrutinib. Methods and materials: In an effort to elucidate the link between BCR signaling and cell adhesion phenotype, we first characterized ibrutinib sensitive and resistant mantle cell lymphoma (MCL) cell lines. We measured cell proliferation and cell growth, and correlated ibrutinib sensitivity with cell adhesion disruption. We then used RNA-sequencing to identify differential pathways between sensitive or resistant cell lines in response to ibrutinib treatment. We validated RNA-Seq findings using cell lines, as well as animal models and human primary MCL tumor tissues and cells. Results: We found that intrinsic sensitivities of MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion gene signatures were simultaneously down-regulated by ibrutinib in ibrutinib-sensitive but not ibrutinib-resistant cell lines. Among the differentially expressed genes in the BCR gene signature, we identified and validated that RAC2, a regulator of cell adhesion, was down-regulated at both RNA and protein levels by ibrutinib only in ibrutinib-sensitive cells. Physical association of RAC2 with BLNK, an early BCR pathway adaptor, was disrupted by ibrutinib uniquely in sensitive cells. RAC2 knockdown with siRNA impaired cell adhesion while RAC2 over-expression rescued ibrutinib-induced reduction in cell adhesion. In a xenograft mouse model, mice treated with ibrutinib demonstrated tumor growth retardation along with down-regulation in RAC2 protein expression. Using immunohistochemical staining, we demonstrated that RAC2 was expressed in ~65% primary MCL tumor tissues with majority of RAC2-positive tumors characterized as being the more aggressive subtypes. Finally, primary MCL cells treated with ibrutinib demonstrated reduced RAC2 that is accompanied by cell adhesion impairment. Conclusions: Our findings uncover a novel cross-talk between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC2. Our study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and new drug development. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; AstraZeneca: Consultancy, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Honoraria, Research Funding.

scholarly journals Venetoclax Resistance in Mantle Cell Lymphoma Is Mediated By BCL-XL and Can be Circumvent By Inhibiting the BH4 Domain of BCL-2

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1507-1507
Author(s):  
Daniela Steinbrecher ◽  
Felix Seyfried ◽  
Eugen Tausch ◽  
Johannes Bloehdorn ◽  
Billy Michael Chelliah Jebaraj ◽  
...  
Keyword(s):  
Cell Viability ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Research Funding ◽  
High Efficiency ◽  
Cell Lymphoma ◽  
Fold Increase ◽  
Dependent Manner ◽  
Independent Manner ◽  
Mantle Cell

Apoptosis is controlled by the expression levels and interplay of pro- and anti-apoptotic BCL-2 family proteins. The specific BCL-2 inhibitor Venetoclax (VEN) showed high efficiency in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Despite its high efficiency in CLL and MCL, refractory disease can develop. BCL-2 mutations have been described to mediate resistance in CLL cases, however these mutations are only found in a proportion of VEN resistant cases and in a fraction of cells. In order to design alternative therapeutic strategies to overcome drug resistance, a better understanding of the mechanisms mediating resistance to VEN is necessary. VEN-resistant (VEN-R) MCL cell lines (MINO and MAVER-1) were generated by chronic exposure to increasing amounts of VEN (up to 3µM). A significant and stable upregulation of BCL-XL mRNA and protein was seen in the MINO and MAVER-1 resistant cell lines (2 and 4 fold increase in mRNA and 2.6 and 4.5 fold increase in protein, respectively). We used BH3 profiling in combination with VEN treatment for 4h to investigate the differences in anti- and pro-apoptotic signaling in parental and VEN-R cell lines. Additionally, sensitivity to VEN was restored upon shRNA-mediated knockdown of BCL-XL. These results confirmed the importance of BCL-XL upregulation in mediating resistance. Furthermore, we did not detect mutations in BCL-2 upon resistance to VEN via targeted NGS, which is in contrast to results obtained in VEN-R CLL patients (Blombery et al., Cancer Discovery 2019 and Tausch et al., Hematologica 2019). However, the results obtained by dynamic BH3-profiling (VEN treatment in combination with BH3 Profiling) suggest that increase in BCL-XL is most likely not the only alteration necessary to render cells resistant to VEN. In addition, reduced activation of pro-apoptotic proteins like BAX and BAK might contribute to resistance to VEN. In order, to investigate if VEN resistance can be overcome by drug mediated inhibition of BCL-XL we used different therapeutic approaches. Combinational treatment with the BCL-XL inhibitor A-1331852 and VEN or the single treatment with Navitoclax, a combined inhibitor of BCL-2, BCL-W and BCL-XL for 48h reduced cell viability in VEN-R MINO and MAVER-1 cell lines. Furthermore, BDA-366, a BH4 domain BCL-2 inhibitor effectively reduced the cell viability after 48h of treatment in a dose dependent manner in both parental and VEN-R cell lines. The binding of BDA-366 to the anti-apoptotic BCL-2 protein leads to a conformational change into a pro-apoptotic molecule by the exposure of the BH3 domain of the protein. Despite mediating apoptosis in a TP53-independent manner, VEN treatment in CLL has been associated with inferior outcome in the presence of TP53 aberrations. In order to address the role of TP53 dysfunction in mediating resistance to VEN, we generated p53 knock out cell lines (N=2) by CRISPR/Cas9 gene editing. This significantly decreased the sensitivity to VEN compared to p53 WT cell lines. Additionally, the sensitivity to BDA-366 was significantly reduced upon knockout of p53, suggesting an interference of p53 downstream of BCL-2. Overall, VEN resistance is mediated by a permanent increase in BCL-XL mRNA and protein level in MCL. Importantly, BDA-366, which converts the anti-apoptotic BCL-2 molecule into a BAX-like death molecule, could be a potential alternative treatment strategy for BCL-2 dependent cancers even when resistant to VEN. Despite mediating apoptosis in a p53 independent manner, VEN seems to be less effective in p53 deficient cells, underlining the importance of further investigations of treatment combinations in these groups. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau. Schneider:Celgene: Other: travel grant.

Activity of Rituximab and Ofatumumab Against Mantle Cell Lymphoma (MCL) in Vitro in MCL Cell Lines and Ex Vivo in Tumor Cells Derived From Patient Tumor Samples

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4972-4972
Author(s):  
Matthew J. Barth ◽  
Gopichand Pendurty ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
John Gibbs ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Clinical Investigations ◽  
Mantle Cell ◽  
Anti Cd20

Abstract Abstract 4972 Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that frequently presents with advanced stage disease. The addition of rituximab, a monoclonal anti-CD20 antibody, to high dose chemotherapy regimens often followed by stem cell transplant has improved outcomes, but survival still remains low at 3–5 years. Novel agents are needed to improve outcomes in MCL. Ofatumumab is a fully human anti-CD20 monoclonal antibody directed against a novel epitope on the CD20 antigen. Ofatumumab has been shown to be more potent than rituximab against B-NHL cells in pre-clinical investigations. Ofatumumab is FDA approved for the treatment of CLL that is fludarabine and alemtuzumab refractory or with bulky disease resistant to fludarabine and is being investigated in clinical trials in NHL. In order to characterize the activity of ofatumumab against MCL, we performed pre-clinical investigations into the activity of ofatumumab against MCL cell lines and primary MCL tumor cells derived from patient tumor samples (n=2). Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in the MCL cell lines Mino, Jeko, Rec-1 and Z-138 to demonstrate sensitivity to rituximab and ofatumumab. Lymphoma cells were labeled with 51Cr prior to incubation with rituximab or ofatumumab at 10ug/mL plus human serum or effector cells (efector:target ratio of 20:1). 51Cr-release was measured and the percentage of lysis was calculated. Patient tumor cells were isolated from tumor biopsy samples by MACS sorting (negative selection). Patient tumor cells were incubated with ofatumumab or rituximab at 10ug/mL in the presence of human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Means were compared using a t-test. Expression of CD20 and the complement inhibitory proteins (CIPs) CD55 and CD59 in MCL cell lines were determined by flow cytometry and compared to the rituximab-sensitive cell line Raji and the rituximab-resistant cell line Raji 4RH. Surface density of CD20, CD55 and CD59 were determined by Imagestream analysis. Western blot was performed to measure total CD20 protein expression. Ofatumumab induced significantly higher levels of cell lysis compared to rituximab in CDC assays of all MCL cell lines tested (Mino: 65.9% vs 0.5%; JeKo 43.9% vs 13.3%; REC-1 25.4% vs 4.7%; Z-138: 56.4% vs 0.65%; all p-values <0.05). The ADCC assays showed a similar degree of lysis with ofatumumab when compared to rituximab in all cell lines tested. In primary tumor cells, ofatumumab and rituximab demonstrated similar levels of decreased cell viability following 48 hours of antibody exposure. MCL cell lines demonstrated similar expression of surface and total CD20 when compared to the rituximab-sensitive B-NHL Raji cell line. CIP expression was increased in all MCL cell lines compared to Raji cells and was similar to the rituximab-resistant Raji 4RH cell line. Our data suggest ofatumumab is more potent than rituximab against MCL cells in vitro and retains CDC activity despite high expression levels of CIPs. This increased activity was not seen in patient tumor samples; however we were limited by the number of available patient samples. In vivo experiments investigating the activity of ofatumumab in a SCID mouse MCL xenograft model and investigations into the activity of ofatumumab in MCL cells in combination with cytotoxic agents and novel small molecule inhibitors are ongoing. Disclosures: Czuczman: Genmab: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding.

scholarly journals Overcoming the Supportive Stroma-Induced Proliferation in Waldenstrom's Macroglobulinemia By Selective Inhibition of the FGF/FGF-Receptor Axis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1822-1822
Author(s):  
Cinzia Federico ◽  
Antonio Sacco ◽  
Katia Todoerti ◽  
Arianna Giacomini ◽  
Gaia C Ghedini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Significant Inhibition ◽  
Advisory Committees ◽  
Dose Dependent

The human fibroblast growth factor receptor (FGF-R) family plays an essential role in a wide range of cellular processes, such as cell growth, proliferation, differentiation, migration and survival. It has been reported that FGF-Rs are expressed in hematopoietic cells; and FGF/FGFR signaling deregulation is largely involved in hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb due to disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGF-R system in FGF-dependent WM cell lines by using an anti-pan FGF trap molecule (NSC12), responsible for FGF/FGF-R blocking. We first interrogated the GSE9656 dataset in order to confirm the expression of FGFs and FGF-Rs in WM cells, demonstrating an enrichment of several FGF- and FGF-R-isoforms in primary WM patients' derived tumor cells compared to the normal cellular counterpart (P<0.05); and demonstrated the ability of NSC12 to inhibit FGF-secretion within the conditioned media of NCS12-treated WM cells, as shown by ELISA. Wide-transcriptome profiling of NSC12-treated WM cells (BCWM.1; MWCL1) revealed a significant inhibition of Myc-target related genes, coupled with silencing of genes involved in cell cycle progression, cell proliferation, PI3K-AKT-mTOR signaling, oxidative phosphorylation (Hallmark; FDR<0.25; P<0.05). This prompted us to evaluate the anti-tumor functional sequelae exerted by NSC12 in WM cells: NSC12 induced significant inhibition of WM cell growth (BCWM1 and WMCL1) in a dose-dependent fashion (0.1-10μM; IC50 ~3μM), even in the presence of bone marrow microenvironment. In addition, a significant effect was also observed in primary tumor cells from WM patients; while no effect was observed on healthy donor-derived peripheral blood mononuclear cells. The growth inhibitory effect was associated with induction of apoptotic cell death, caspase activation and PARP cleavage, as demonstrated by flow cytometry and western blot, respectively. Moreover, we also observed a NSC12 dose-dependent increase of mitochondrial reactive oxigen species (ROS), at protein level. Cell cycle analysis revealed a reduction of the S-phase and increase of G0/G1 phase. Mechanistically, NSC12 targeted WM cells by inhibiting MAPK, JAK/STAT3 and PI3K-Akt pathways known to be FGFRs-activated signaling cascades. Importantly, the same effect was maintained in WM cells even in the presence of the supporting BM microenvironment. Functional studies demonstrated the ability of NSC12 to impair the adhesion of both cell lines to the supportive primary bone marrow stromal cells, in vitro. NCS12-dependnet anti-WM activity was also tested in combination with bortezomib, carfilzomib, everolimus and ibrutinib: the combinatory treatment (48h) resulted in a more significant dose-dependent inhibition of WM cell survival and proliferation (P<0.05), thus suggesting the rational for combining FGF-blockade with proteasome-, mTOR-, or BTK-inhibitors. In vivo studies are being performed, in order to further corroborate the anti-WM activity of NSC12 using WM animal models. Disclosures Ronca: Associazione Italiana per la Ricerca sul Canctro (AIRC): Research Funding. Rossi:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Roccaro:AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding.

CUDC-907: An Oral HDAC/PI3K Dual Inhibitor with Strong Preclinical Efficacy in MCL Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4183-4183 ◽  
Author(s):  
Jordan N Boyle ◽  
Caroline R Kim ◽  
Hui Guo ◽  
Taylor Bell ◽  
Shengjian Huang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Dual Inhibitor ◽  
Pdx Model ◽  
Ic50 Values ◽  
Dose Dependent

Abstract Introduction Mantle cell lymphoma (MCL) is an aggressive lymphoma with elevated B-cell receptor activity. Ibrutinib (IBN), a Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have a response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN-resistant lymphomas, new therapies are needed. It is suspected that upregulation of the PI3K-Akt-mTOR pathway allows survival in the presence of IBN. CUDC-907 is a next generation PI3K and HDAC dual-inhibitor currently in phase II clinical trials. Our objective is to investigate the effects of CUDC-907 in IBN-resistant MCL cells in vitro and in PDX model. Methods MCL cells were seeded at 10,000 cells per well in 96-well plate and were treated with various doses of compounds at the following concentrations: CUDC-907/Ibrutinib 0.015, 0.05, 0.15, 0.5, 1.5, 5, and 15 uM. Cell viability was tested by CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation. Next, MCL cells were incubated with IBN at varying doses (0.39 uM, 1.56 uM, 6.25 uM), CUDC-907 (0.39 uM, 1.56 uM, 6.25 uM), and IBN+CUDC-907 (0.39 uM, 1.56 M, 6.25 uM) for 24 hours. Apoptosis was detected by Annexin V-binding assay. In patient derived xenograft (PDX) model: CUDC-907 was administered at a dose of 50 mg/kg in ibrutinib-resistant MCL-bearing PDX mice daily. Tumor volumes were measured as the length X width2 X 0.5 weekly. Toxicity was also observed every week. Mice were sacrificed once diameter of tumor mass reached 15 mm size. Results CUDC-907 inhibited the growth of both ibrutinib-sensitive and resistant MCL cells in vitro. Sensitive cell lines include: Rec-1, Mino, and JVM-13 with IC50 values of 1.1 nM, 1.0 nM, and 5 nM respectively. Resistant cell lines include: Granta-519, Maver-1, and Z-138 with IC50 values of 2 nM, 3 nM, and 1.5 nM respectively. All tested cell lines were more sensitive to CUDC-907 than to ibrutinib. In addition, combination treatments of CUDC-907 and IBN increased cell death in comparison to single agent treatments. Next, one pair of MCL cell lines, Jeko-1 (sensitive to IBN) and Jeko-R (resistant to IBN) were treated for 24 hours with varying doses of CUDC-907 or ibrutinib either as single agent inhibitors or in combination therapies. The results demonstrated that CUDC-907 induced apoptosis in both Jeko-1 and Jeko-R cell lines in a dose-dependent manner. Combination therapies increased cell death in a dose-dependent manner as well. In PDX model, tumor volume in treated mice of ibrutinib-resistant PDX decreased significantly compared with vehicle control (pvalue = 0.032). Control mice also weighed considerably more than treated mice (p value = 0.073). Common toxicities included a decrease in body mass for first 28 days of treatment. Conclusion CUDC-907, a dual inhibitor of PI3K-Akt-mTOR and HDAC, inhibits tumor growth of ibrutinib-resistant MCL in vitro and in PDX model. It would be a potential drug for the patients with ibrutinib-resistant/relapsed MCL. Disclosures Wang: Celgene: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Research Funding; Juno Therapeutics: Research Funding; Asana BioSciences: Research Funding.

scholarly journals Simultaneous Inhibition of BCL-2 and PI3K Signaling Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2950-2950
Author(s):  
Haige Ye ◽  
Shengjian Huang ◽  
Dayoung Jung ◽  
Changying Jiang ◽  
Krystle Nomie ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Tumor Burden ◽  
Pi3k Signaling ◽  
Advisory Committees ◽  
Apoptotic Pathway ◽  
Mantle Cell ◽  
Ibrutinib Resistance

Abstract Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) has achieved a 68% overall response rate in relapsed/refractory MCL patients (Wang et al., NEJM, 2013). However, the vast majority of MCL patients experience disease progression; therefore, novel therapies to overcome ibrutinib resistance are urgently needed. Ibrutinib resistance is associated with the dysregulation of alternative signaling pathways such as the apoptotic pathway and PI3K/AKT signaling; therefore, combinatorial therapeutic strategies may prove fruitful in overcoming ibrutinib resistance via the blockade of these compensatory pathways. Based on the upregulation of the PI3K signaling pathway and the anti-apoptotic pathway in ibrutinib resistance, we co-inhibited both pathways with the dual PI3K-delta and -gamma inhibitor duvelisib and the BCL-2 inhibitor venetoclax to assess the ability of this combination to overcome ibrutinib resistance in MCL. Methods: Cell viability and apoptosis assays were conducted to assess the effects of venetoclax and duvelisib on 4 ibrutinib-resistant cell lines (Jeko BTK KD, Jeko-R, Z-138 and Maver-1) and 4 primary patient samples. We also confirmed the synergistic effect of this combination on two ibrutinib-resistant cell lines (Jeko BTK KD and Z-138) by western blotting. Aberrant protein expression between single agents and drug combination in Jeko BTK KD cells was detected using Reverse Phase Protein Array (RPPA) analysis with confirmation by western blotting. Cell migration of Jeko BTK KD cells was assessed. A Jeko-1 cell xenograft (which is resistant to ibrutinib in vivo) was established in NSG mice, and drug testing was performed in this model with tumor burden assessed viain vivo imaging. Results: We observed synergistic activity with the combination of venetoclax and duvelisib in 4 ibrutinib-resistant cell lines (Jeko BTK KD, 0.36; Jeko-R, 0.47; Z-138, 0.66; Maver-1, 0.41) and in 4 ibrutinib-resistant patient samples (PT1, 0.14; PT2, 0.43; PT3, 0.52; PT4, 0.41) by calculating the synergistic index (Ki value). We confirmed the synergistic effects of this combination by observing apoptosis at 72 hours post-treatment in Jeko BTK KD and Z-138 cells. Moreover, the combination of venetoclax and duvelisib synergistically reduced the cell migration of Jeko BTK KD cells. RPPA analysis of Z-138 MCL cells post-treatment demonstrated that multiple pathways, including the apoptotic pathway as well as the PI3K/AKT and BCR signaling pathways, were synergistically altered by venetoclax and duvelisib. Moreover, venetoclax and duvelisb synergistically reduced tumor burden in a Jeko-1 cell xenograft model resistant to ibrutinib. Conclusion: PI3K signaling and BCL-2-related pathways are activated in ibrutinib-resistant MCL cells, and targeting these pathways using a combinatorial approach may potentially overcome ibrutinib resistance. Disclosures Wang: AstraZeneca: Consultancy, Research Funding; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; MoreHealth: Consultancy; Juno: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria.

scholarly journals Characterization of Mechanisms Underlying Acquired Venetoclax-Resistance in Mantle Cell Lymphoma: BDA-366 As a Potential Treatment Option

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1580-1580
Author(s):  
Daniela Steinbrecher ◽  
Felix Seyfried ◽  
Johannes Bloehdorn ◽  
Billy Michael Chelliah Jebaraj ◽  
Lüder Hinrich Meyer ◽  
...  
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Advisory Committees ◽  
Apoptotic Protein ◽  
Mantle Cell ◽  
Resistant Cells

Abstract In many cancers the equilibrium of pro- versus anti-apoptotic BCL-2 proteins is deregulated. BCL-2 inhibitors like Venetoclax (VEN) have been shown to be highly active drugs in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Despite being highly efficient in cell killing, resistance to VEN can be acquired over time. In addition to understanding the underlying mechanisms of resistance to VEN it is important to identify additional treatment options. BDA-366 is a BCL-2 inhibitor with a different mode of action than the BH3 mimetic VEN. BDA-366 acts by inhibiting the BH4 domain and thereby inducing a conversion of anti-apoptotic BCL-2 into a pro-apoptotic protein. BDA-366 showed high effectivity in inducing apoptosis in CLL cells, in primary as well as in cell lines, while all of the CLL cell lines (n=7) tested were resistant to VEN. Furthermore all of the MCL cell lines (n=5) tested were sensitive to the treatment with BDA-366 while only a subset (3 out of 5) responded to treatment with VEN. In order to investigate whether BDA-366 would be a treatment option for VEN-resistant patients, we generated VEN-resistant MCL cell lines (MINO and MAVER-1) by chronic exposure to the drug. In the resistant cell lines, BCL-2 protein levels were not deregulated. In variance to previous reports in diffuse large B cell lymphoma (DLBCL) (Choudhary et al, Cell Death Dis 2015), resistance in MCL cell lines was not mediated by MCL-1 upregulation. In VEN-resistant MINO cells, MCL-1 expression was similar to the parental cells, while MCL-1 was significantly downregulated in VEN-resistant MAVER-1 cells. In contrast, VEN-resistant MCL cell lines showed BCL-XL upregulation as compared to parental cells, which is in line with results obtained in DLBCL (Choudhary et al, Cell Death Dis 2015). Furthermore, dynamic BH3 profiling validated a dependency on BCL-XL in resistant cells and confirmed that resistance was not mediated by MCL-1. The significance of BCL-XL in mediating resistance to VEN was underlined by additional experiments using navitoclax. In contrast to VEN, navitoclax inhibits BCL-2, BCL-XL and BCL-W and was sufficient to induce apoptosis in both parental and resistant cells. In contrast to the BH3 domain inhibitor VEN, the BCL-2 inhibitor BDA-366 acts by converting BCL-2 into a pro-apoptotic molecule. BDA-366 efficiently induced dose dependent apoptosis in VEN-resistant cells. MINO as well as MINO VEN-resistant cells showed the same sensitivity to BDA-366 while VEN-resistant MAVER-1 cells showed reduced sensitivity to BDA-366 as compared to the parental cells. However, with increased BDA-366 concentrations efficient cell killing was achieved in the VEN-resistant cell lines Overall, these results suggest that VEN-resistance is mostly mediated by permanent upregulation of BCL-XL. BCL-2 levels are not deregulated upon development of resistance to VEN. The inhibition of the BH4 domain and thereby converting BCL-2 into a pro-apoptotic protein proved to be a promising therapeutic option even in cancers with acquired resistance to VEN. Disclosures Döhner: Pfizer: Research Funding; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Pfizer: Research Funding; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3714-3714 ◽  
Author(s):  
Lei Wu ◽  
Peter Schafer ◽  
George Muller ◽  
David Stirling ◽  
J. Blake Bartlett
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Dependent Manner ◽  
Activation Markers ◽  
Early Results ◽  
Ifn Γ ◽  
Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Preliminary Data ◽  
Research Funding ◽  
Preclinical Studies ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1844-1844 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle Kuhne ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Mouse Models ◽  
Research Funding ◽  
Therapeutic Agents ◽  
Dependent Manner ◽  
Apoptotic Pathways ◽  
Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

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