scholarly journals Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 952-952 ◽  
Author(s):  
Shannon M Matulis ◽  
Vikas A. Gupta ◽  
Izabelle Brown ◽  
Jonathan J Keats ◽  
Paul Secrist ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Contact ◽  
Advisory Committees ◽  
Highly Sensitive ◽  
Ic50 Values ◽  
Cell Cell

Abstract We and others have previously demonstrated that MM is often dependent on MCL1 or co-dependent on MCL1 and BCLXL or BCL2 for survival. Therefore, drug development targeting MCL1 has been a top priority. Here we report on AZD5991, a specific small molecule inhibitor of MCL1. We treated 17 MM cell lines with increasing concentrations of AZD5991 for 24 h and measured Annexin V staining to determine the IC50s. Nine of the cell lines tested were highly sensitive to AZD5991 with IC50 values below 100 nM, 6 lines exhibited an intermediate sensitivity (IC50 100-1000 nM), and only 2 cell lines tested were resistant (IC50 >1000 nM). Six of the highly sensitive lines are t(11;14) and sensitive to venetoclax suggesting co-dependence on BCL2 and MCL1 for survival. We also determined the effect of the bone marrow microenvironment on the response of MM cell lines to AZD5991. We reported that IL-6 protects MM cell lines and patient samples from apoptosis by making the cells more MCL1 dependent. Based on this, we predicted IL-6 would have little to no effect on AZD5991-induced cell death. We treated 12 cell lines with AZD5991 in the presence of 1 ng/mL IL-6 or 10% Hs5 conditioned medium (CM) for 24 h and found that only 3/12 and 2/12 lines were protected from apoptosis in the presence of IL-6 and CM, respectively. Interestingly, when co-cultured with the stromal cell line Hs5, 7/11 lines tested were protected from AZD5991-induced cell death, suggesting cell-cell contact is influencing the response. This is in contrast to ABT-737 and venetoclax where cell-cell contact provided no additional protection than CM. Mechanistically apoptosis induced via MCL1 inhibition is not dependent on BIM expression as is the case with BCLXL and BCL2 inhibition. KMS26 and LP1 MM cell lines contain a bi-allelic deletion of BIM and we have reported their resistance to ABT-737. However, both cell lines respond to AZD5991 with IC50 values in an intermediate sensitivity range. Co-immunoprecipitation (CoIP) studies were employed to determine the protein bound to MCL1 that could be promoting apoptosis upon release. We found NOXA and BAK bound in KMS26 and LP1 and both were released from MCL1 in response to AZD5991. Additionally, CoIPs performed on cell lines expressing BIM showed NOXA, BIM, and BAK bound to MCL1 and released following treatment. To further investigate we used CRISPR-cas9 to generate MM cell lines lacking expression of NOXA, BAK, BAX, or BIM. In KMS26 and LP1, deletion of NOXA and BAX had little effect on AZD5991-induced cell death while the BAK deletion significantly inhibited apoptosis in both cell lines. Similar results were observed in the BIM expressing cell line OCI-My5, with no protection from AZD5991-induced apoptosis in the NOXA and BAX edited lines, significant protection in the BAK-deleted line, and an intermediate degree of protection in the BIM knockout line. In KMS18, BIM deficiency had a minimal effect on apoptosis following MCL1 inhibition, however both BAX and BAK were required for AZD-induced cell death. Additionally, we have tested 41 samples from 37 patients for sensitivity to AZD5991. Samples were treated with increasing concentrations to determine IC50 values in the same manner as the MM cell lines. The samples segregated into 4 groups based on IC50. The most sensitive group (N=3) had an IC50 below 10 nM. The largest group had an IC50 range of 50-114 nM (N=26). The last two cohorts were more resistant with a range of 500-916 nM (N=10) and 2 samples with an IC50 over 1300 nM. Since MCL1 is on 1q21, a frequently amplified region in MM, we determined if 1q21 gain was associated with sensitivity. For the 35 samples where FISH data were available, 18 had 1q21 gains by FISH while 17 were negative. There is a trend for the 1q21 gain cohort to be more sensitive (P=0.0573), with only 2/18 having an IC50 above 109 nM. In contrast for the 1q21 negative 7/17 were in the resistant groups. Thus 1q21 may be a marker of sensitivity to MCL1 inhibitors. The data reported here demonstrate that AZD5991 is effective at inducing apoptosis in MM and can overcome soluble microenvironment resistance factors that influence the response to venetoclax. This appears to be due to differential requirements for pro-apoptotic factors for BCL2 and MCL1 inhibition and suggests an underappreciated complexity in the role of BCL2 and MCL1 in cell survival. Finally these findings also suggest that 1q21 gain may be a marker for AZD5991 sensitivity. A clinical trial is currently ongoing in myeloma. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Neri:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Heffner:Pharmacyclics: Research Funding; Genentech: Research Funding; ADC Therapeutics: Research Funding; Kite Pharma: Research Funding. Lonial:Amgen: Research Funding. Nooka:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

The Interaction of Bortezomib with P-Gp, MRP-1 and BCRP Drug Transporters: Implications for Therapeutic Applications of Bortezomib in Advanced Multiple Myeloma and Other Neoplasias.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1729-1729
Author(s):  
Melissa G Ooi ◽  
Robert O'Connor ◽  
Jana Jakubikova ◽  
Justine Meiller ◽  
Steffen Klippel ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Significant Activity ◽  
Cancer Resistance ◽  
Advisory Committees ◽  
Bortezomib Treatment

Abstract Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

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.

scholarly journals Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4356-4356
Author(s):  
John S Manavalan ◽  
Ipsita Pal ◽  
Aidan Pursley ◽  
George A. Ward ◽  
Tomoko Smyth ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Tnf Alpha ◽  
Sensitive Cell ◽  
Advisory Committees

Abstract Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). Figure 1 Figure 1. Disclosures Smyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

scholarly journals CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3199-3199 ◽  
Author(s):  
Subhashis Sarkar ◽  
Sachin Chauhan ◽  
Arwen Stikvoort ◽  
Alessandro Natoni ◽  
John Daly ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Cd38 Expression ◽  
Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Multiomic Mapping of Copy Number and Structural Variation on Chromosome 1 (Chr1) Highlights Multiple Recurrent Disease Drivers

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 721-721
Author(s):  
Patrick Blaney ◽  
Eileen M Boyle ◽  
Yubao Wang ◽  
Hussein Ghamlouch ◽  
Jinyoung Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Copy Number ◽  
Research Funding ◽  
Chromosome 1 ◽  
Structural Variants ◽  
Advisory Committees

Abstract Introduction Copy number abnormalities (CNA) and structural variants (SV) are crucial to driving cancer progression and in multiple myeloma (MM). Chr1 CNA are seen in up to 40% of cases and associate with poor prognosis. Variants include deletions, gains, translocations and complex SV events such as chromothripsis (CT), chromoplexy (CP) and templated insertions (TI) which result in aberrant transcriptional patterns. Abnormal expression of genes on chr1 lead to the adverse clinical outcome and studies focussed on 1p12, 1p32.3 and 1q12-21 identified potential causal genes including TENT5C, CDKN2C, CKS1B, PDZK1, BCL9, ANP32E, ILF2, ADAR, MDM2 and MCL1 but none fully explain the clinical behavior. To address this deficiency and to relate chromatin structure to gene deregulation we present a multiomic bioinformatic analysis of SV, CNA, mutation and expression changes in relation to the chromatin structure of chr1. Methods We analysed data derived from 1,154 CoMMpass trial patients. We analyzed 972 NDMM patients with whole exome for mutations, and 752 whole genomes for copy number, translocations, complex rearrangements such as CP, CT and TI as previously described. Using GISTIC 2.0, we identified hotspots of CNA. This information was then analyzed in conjunction to the RNA-seq data derived from 643 patients to determine the aberrant transcriptional landscape of chr1. Using HiC data derived from U266 MM cell line, we associated these changes with TAD structures, A/B compartments, and histone marks along chr1, to gene expression changes, and recurrent SV. Using the cell line dependency map for CRISPR knockdown of the gene set on chr1 derived from 20 MM cell lines we related cell viability to chr1 copy number status. Results We identified 7 hotspots of deletion, 9 of gain, 3 of CT and 2 of templated-insertion across chr1. We mapped these regions to epigenetic plots and show that gained regions are hypomethylated compared to the rest of chr1 (Wilcoxon, p=0.0002). Overall 69% of gain(1q) and 45% of the non-gained hotspots were in A compartments (χ 2=11, p=0.0009) and had an overall higher compartment score (p=0.01).The recurrent regions of loss on 1p confirm the clinical relevance of this region. The critical importance of TENT5C, CDKN2C and RPL5 is identified by the impact of deletion, mutation and the rearrangement of superenhancers. Further this convergence of multiple oncogeneic mechanisms to a single locus points to a number of novel candidate drivers including FUB1 and NTRK1.We provide important new information on 1q21.1-1q25.2 encompassing 145-180Mb a transcriptionally dense region containing 6 GISTIC 2.0 hotspots of gain (G2-G7). The hotspots occur within TAD structures that correlate upregulation of known drivers listed above and also identified novel potential upregulated drivers including POU2F1, a transcription factor, CREG1, an adenovirus E1A protein that both activates and represses gene expression promoting proliferation and inhibiting differentiation (G6) and BTG2 a G1/S transition regulator (G8). These data for copy number gain provides strong evidence for the prognostic relevance of of multiple drivers within deregulated TADs rather than single candidate genes. It also highlights the importance of the chromatin structure of Chr1 in the generation of these events.Using dependency map CRISPR data we identified 320 essential genes for at least one cell line (>1). A common set of 31 genes were identified including 3 proteasome subunits (PSMA5, PSMB2, PSMB4), three regulators of ubiquitin-protein transferase activity (RPL5, RPL11, CDC20), splicing (SF3B4, SF3A3, SFPQ, RNPC3, SRNPE, PRPF38A, PRPF38B) and DTL. A common dependency for 1q+ or 1p- was not identified but a number of dependencies were identified in more than one cell line including UQCRH, SLCA1, CLSPN in 1p- cell lines and IPO9, PPIAL4G, and MRPS2 in 1q+. Conclusion We present an elegant anatomic map of chr1 at the genetic and epigenetic levels providing an unprecedented level of resolution for the relationships of structural variants to epigenetic, expression and mutation status. The analysis highlights the importance of active chromatin in gene deregulation by SV and CNA where the importance of multiple gene deregulation within TAD structures is critical to MM pathogenesis. The implications are that we could improve prognostic assignment and identify new targets for therapy by further characterizing these relationships. Figure 1 Figure 1. Disclosures Braunstein: Jansen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Davies: Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1741-1741
Author(s):  
Steffen Klippel ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Melissa G. Ooi ◽  
Douglas McMillin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cancer Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Energy Production ◽  
Advisory Committees ◽  
Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1470-1470
Author(s):  
Jueqiong Wang ◽  
Chung Hoow Kok ◽  
Richard J. D'Andrea ◽  
Timothy P. Hughes ◽  
Deborah L. White
Keyword(s):  
Cell Death ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
K562 Cells ◽  
Vehicle Control ◽  
Peroxisome Proliferator ◽  
Advisory Committees ◽  
Peroxisome Proliferator Activated Receptor

Abstract Introduction The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. Methods OA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. Results A significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05). Conclusion Ligand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy. 1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures: Hughes: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

scholarly journals A CK1δ/CK1ε-to-Wnt/β-Catenin Circuit Is a Therapeutic Vulnerability in Primary and Drug Resistant Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2094-2094
Author(s):  
Karen L. Burger ◽  
Mark B Meads ◽  
Ariosto Silva ◽  
Allison Distler ◽  
Maria Coelho Siqueira Silva ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Wnt Signaling ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Highly Sensitive ◽  
Tumor Types

Abstract Aberrant canonical Wnt/β-catenin signaling has long been known to play a role in cancer development and progression, where Wnt binding provokes nuclear localization of β-catenin, which functions as a coactivator for the TCF/LEF family of transcription factors that induce an oncogenic transcriptional program. Indeed, hallmarks of several tumor types are gain-of-function somatic mutations in β-catenin, and loss-of-function mutations in components of the β-catenin destruction complex, including the scaffold proteins APC and Axin1 and the serine/threonine kinase casein kinase1-α (CK1α) that phosphorylates β-catenin, priming it for destruction by the βTrCP E3 ubiquitin ligase. Interestingly, the CK1α-related kinase CK1δ antagonizes CK1α in Wnt signaling, where CK1δ triggers disassembly of the β-catenin destruction complex by phosphorylation of disheveled-1 (Dvl1). Notably, we have shown that CK1δ is amplified, and is necessary and sufficient, for β-catenin activation and Wnt signaling in some tumor types. Interestingly, lenalidomide resistance in multiple myeloma (MM) cell lines and patients has been shown to correlate with increased expression and activity of Wnt/β-catenin signaling. Here, we report that roughly 40% of MM patients have mutations in the Wnt/β-catenin pathway and that this pathway is active in a large panel of human MM cell lines. Notably, a highly potent and selective in-house CK1δ/CK1ε kinase inhibitor coined SR-3029 rapidly compromises the growth and survival of MM cell lines. Importantly, the anti-MM activity of SR-3029 is augmented in MM cell lines with selected resistance to bortezomib and lenalidomide relative to paired naïve myeloma cells. In concordance with the anti-MM activity of SR-3029, treatment of MM cells with this kinase inhibitor leads to marked reductions in the levels of β-catenin target genes (e.g., MYC, CCND1 and WNT3) and also with the suppression of CK1δ and CK1ε. Further, using an ex vivo platform that accurately quantifies the sensitivity of primary MM samples to agents in a reconstructed tumor microenvironment, we examined CD138-selected patient specimens from 29 patients against a panel of 31 drugs simultaneously. Notably, MM patient samples, including those that are quad-resistant, were highly sensitive to SR-3029 with a mean LD50 of 300nM and a range between 30nM and 990nM. Indeed, SR-3029 was by far the most potent kinase inhibitor assessed in this platform, where its mean LD50 is an order of magnitude more potent than all other kinase inhibitors included in the screen. Finally, using the well-established 5TGM1/Kal-Ridge (C57B6/KaLwRijHsd) syngeneic mouse model of multiple myeloma, we show that tumors derived from 5TGM1 cells, which are highly sensitive to SR-3029 ex vivo, are also sensitive to CK1δ/CK1ε inhibition in vivo, where the SR-3029 treated cohort of animals demonstrated decreased tumor burden as assessed by IgG2b levels and imaging, and by significantly improved survival relative tovehicle treated recipients. Collectively, these findings demonstrate that SR-3029 has potent activity in both naïve and therapy resistant multiple myeloma and establish CK1δ and/or CK1ε as attractive targets for anti-MM therapy. Disclosures Shain: Novartis: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Signal Genetics: Research Funding; Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals BAR-Bodies: B-Cell Receptor Antigens As the Targeting Moiety of Antibodies in Substitution for the Variable Region of Heavy and Light Chains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2940-2940
Author(s):  
Moritz Bewarder ◽  
Lorenz Thurner ◽  
Frank Neumann ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Advisory Committees

Abstract Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: 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; Hoffmann La-Roche: 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; Janssen: 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; GSK: 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; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; 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; AbbVie: 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.

scholarly journals A New Therapeutic Hypothesis: Nonsense-Mediated Decay Is an Exploitable Target in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1937-1937
Author(s):  
Alexander Leeksma ◽  
Ingrid A.M. Derks ◽  
Brett Garrick ◽  
Torsten Trowe ◽  
Aldo Jongejan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Protein Production ◽  
Splicing Factors ◽  
Advisory Committees ◽  
Nonsense Mediated Decay

Abstract Background Nonsense-mediated decay (NMD) is a cellular quality control system that degrades mRNAs containing premature termination codons (PTCs) as well as ~10% of normal mRNAs (Kurosaki and Maquat, 2016). NMD thus prevents translation of misfolded proteins, and potential activation of the unfolded protein response (UPR). Mutations in splicing factors such as SF3B1, SRSF2, U2AF1 and ZRSR2 found in hematological as well as solid tumors, can lead to generation of aberrant mRNAs that contain PTCs. Aberrant splicing patterns in cancer cells can possibly result in increased pressure on the NMD machinery. CC-115, a potent inhibitor of mTOR kinase (TORK) and of DNA-dependent protein kinase, (DNA-PK; Mortensen et al., 2015; Tsuji et al., 2017), is in clinical development for the treatment of solid and hematologic malignancies (Thijssen et al., 2016). Preclinical data revealed an additional target of CC-115 and its differential effect on NMD. Our hypothesis was that a subset of tumor cells, especially hematologic tumors with high protein production and/or splicing factor mutations, would be susceptible to NMD inhibition by CC-115. Methods In total, 141 cell lines were screened for sensitivity to CC-115-mediated inhibition of proliferation and induction of cell death, in comparison to specific inhibition of TORK (CC-223). Isogenic DNA-PK knockout cell lines HCT116/HCT116 DNA-PK-/- and M059K/M059J DNA-PK-/- were treated with CC-115 and CC-223. Activity on NMD in vivo was tested using HCT-116 xenograft tumors treated with Vehicle or CC-115. Dependence on CC-115 sensitivity was determined using CRISPR/Cas9 technology of apoptosis or UPR genes in various MM cell lines. RNA sequencing was used for identification of potential targets in sensitive and resistant cell lines. Results A subset of cancer cell lines underwent cell death at sub-micromolar concentrations of CC-115 due to inhibition of NMD, but this was independent of mutations in splicing factors such as SF3B1. We next focused on MM cells as these generally produce high levels of (immunoglobulin) proteins and are prone to ER stress, and therefore potentially susceptible to NMD inhibition. Indeed, treatment with CC-115 resulted in activation of the UPR independent of TORK and DNA-PK inhibition, and cell death in 11/12 MM cell lines. Activity of CC-115 correlated strongly with cell death by the known ER-stress inducer, thapsigargin. Cell death by CC-115 occurred by the mitochondrial pathway of apoptosis, as it depended on caspase activity and the presence of Bax-Bak. Analysis of RNA sequencing data is ongoing and has indicated potential targets dictating sensitivity to CC-115-mediated cell death. Conclusions We describe that hematologic tumors with high protein production are specifically sensitive to CC-115, a novel and clinically exploitable inhibitor of NMD. This might lead to application in malignancies that depend on NMD to avoid excessive protein stress, such as multiple myeloma. Disclosures Garrick: Celgene: Employment. Trowe:Celgene: Employment. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Eldering:Celgene: Research Funding. Filvaroff:Celgene: Employment.

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