Kinesin Spindle Protein Inhibitor Research Articles

38IN - Kinesin spindle protein inhibitors as novel payload class for ADCs

38IN - Kinesin spindle protein inhibitors as novel payload class for ADCs

Cobalt-Catalyzed Asymmetric Allylation of Cyclic Ketimines.

A CoII /Box-catalyzed [Box=bis(oxazoline)] enantioselective addition of potassium allyltrifluoroborate to cyclic ketimines was developed, providing the corresponding chiral α-tertiary amines in high yields and with good enantioselectivity values. Alkoxycarbonyl- and alkyl-substituted saccharin-derived ketimines are suitable substrates for this allylation reaction. The product can be converted to complex molecules over several simple steps, including a precursor of MK-0371, which is a kinesin spindle protein inhibitor. In addition, this catalytic system showed a strong positive nonlinear effect.

Ligand- and structure-based in silico studies to identify kinesin spindle protein (KSP) inhibitors as potential anticancer agents

Kinesin spindle protein (KSP) belongs to the kinesin superfamily of microtubule-based motor proteins. KSP is responsible for the establishment of the bipolar mitotic spindle which mediates cell division. Inhibition of KSP expedites the blockade of the normal cell cycle during mitosis through the generation of monoastral MT arrays that finally cause apoptotic cell death. As KSP is highly expressed in proliferating/cancer cells, it has gained considerable attention as a potential drug target for cancer chemotherapy. Therefore, this study envisaged to design novel KSP inhibitors by employing computational techniques/tools such as pharmacophore modelling, virtual database screening, molecular docking and molecular dynamics. Initially, the pharmacophore models were generated from the data-set of highly potent KSP inhibitors and the pharmacophore models were validated against in house test set ligands. The validated pharmacophore model was then taken for database screening (Maybridge and ChemBridge) to yield hits, which were further filtered for their drug-likeliness. The potential hits retrieved from virtual database screening were docked using CDOCKER to identify the ligand binding landscape. The top-ranked hits obtained from molecular docking were progressed to molecular dynamics (AMBER) simulations to deduce the ligand binding affinity. This study identified MB-41570 and CB-10358 as potential hits and evaluated these experimentally using in vitro KSP ATPase inhibition assays.

Current and New Therapeutic Strategies for Relapsed and Refractory Multiple Myeloma: An Update.

Although survival of multiple myeloma patients has at least doubled during recent years, most patients eventually relapse, and treatment at this stage may be particularly complex. At the time of relapse, the use of alternative drugs to those given upfront is current practice. However, many new options are currently available for the treatment of relapsed multiple myeloma, including recently approved drugs, such as the second- and third-generation proteasome inhibitors carfilzomib and ixazomib, the immunomodulatory agent pomalidomide, the monoclonal antibodies daratumumab and elotuzumab and the histone deacetylase inhibitor panobinostat, but also new targeted agents are under active investigation (e.g. signal transduction modulators, kinesin spindle protein inhibitors, and inhibitors of NF-kB, MAPK, AKT). We here describe a new paradigm for the treatment of relapsed multiple myeloma. The final goal should be finding a balance among efficacy, toxicity, and cost and, at the end of the road, achieving long-lasting control of the disease and eventually even cure in a subset of patients.

The kinesin spindle protein inhibitor filanesib enhances the activity of pomalidomide and dexamethasone in multiple myeloma.

Kinesin spindle protein inhibition is known to be an effective therapeutic approach in several malignancies. Filanesib (ARRY-520), an inhibitor of this protein, has demonstrated activity in heavily pre-treated multiple myeloma patients. The aim of the work herein was to investigate the activity of filanesib in combination with pomalidomide plus dexamethasone backbone, and the mechanisms underlying the potential synergistic effect. The ability of filanesib to enhance the activity of pomalidomide plus dexamethasone was studied in several in vitro and in vivo models. Mechanisms of this synergistic combination were dissected by gene expression profiling, immunostaining, cell cycle and short interfering ribonucleic acid studies. Filanesib showed in vitro, ex vivo, and in vivo synergy with pomalidomide plus dexamethasone treatment. Importantly, the in vivo synergy observed in this combination was more evident in large, highly proliferative tumors, and was shown to be mediated by the impairment of mitosis transcriptional control, an increase in monopolar spindles, cell cycle arrest and the induction of apoptosis in cells in proliferative phases. In addition, the triple combination increased the activation of the proapoptotic protein BAX, which has previously been associated with sensitivity to filanesib, and could potentially be used as a predictive biomarker of response to this combination. Our results provide preclinical evidence for the potential benefit of the combination of filanesib with pomalidomide and dexamethasone, and supported the initiation of a recently activated trial being conducted by the Spanish Myeloma group which is investigating this combination in relapsed myeloma patients.

A Phase 1 and 2 study of Filanesib alone and in combination with low‐dose dexamethasone in relapsed/refractory multiple myeloma

Filanesib (ARRY-520) is a highly selective inhibitor of kinesin spindle protein, which has demonstrated preclinical antimyeloma activity. This open-label Phase 1/2 study determined the maximum tolerated dose of Filanesib administered on Days 1 and 2 of 14-Day Cycles in patients with multiple myeloma (MM) and included expansion cohorts with and without dexamethasone (40 mg/week). Patients in the dose-escalation (N = 31) and Phase 2 single-agent (N = 32) cohorts had received prior bortezomib as well as prior thalidomide and/or lenalidomide. Patients in the Phase 2 Filanesib plus dexamethasone cohort (N = 55) had received prior alkylator therapy and had disease refractory to lenalidomide, bortezomib, and dexamethasone. Prophylactic filgrastim was incorporated during dose escalation and was used throughout Phase 2. Patients in each cohort had received a median of ≥6 prior therapies. The most common dose-limiting toxicities were febrile neutropenia and mucosal inflammation. In Phase 2, Grade 3 and 4 cytopenias were reported in approximately 50% of patients. Nonhematologic toxicities were infrequent. Phase 2 response rates (partial responses or better) were 16% (single agent) and 15% (Filanesib plus dexamethasone). All responding patients had low baseline levels of α1-acid glycoprotein, a potential selective biomarker. Filanesib 1.50 mg/m2 /day administered with prophylactic filgrastim has a manageable safety profile and encouraging activity in heavily pretreated patients This study is registered at www.clinicaltrials.gov as NCT00821249. Cancer 2017;123:4617-4630. © 2017 American Cancer Society.

Abstract 3234: Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class

Abstract The number of cytotoxic payload classes with different modes-of-action which have been successfully employed in antibody-drug conjugates (ADC) is still rather limited. So far, only ADCs with microtubule inhibitors, DNA binding payloads or topoisomerase I inhibitors have been advanced into clinical testing. To this end, the identification of ADC payload classes with a novel mode of action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of kinesin spindle protein (KSP/Eg5) have generated interest due to their high antitumor potency. However, transferring the preclinical potency of small molecule KSP inhibitors (KSPis) into highly efficient clinical regimens with a sufficient therapeutic window has remained challenging. We have investigated a new pyrrole subclass of KSPis which showed subnanomolar potency against a large panel of tumor cell lines for their utility as a novel payload class in ADCs. Towards this goal different attachment sites for linkers have been explored in the KSPi molecule which were found compatible with cleavable and/or non-cleavable linkers. Subnanomolar potency and selectivity of ADCs with antibodies targeting either HER2, EGFR or TWEAKR could be demonstrated in vitro. For selected ADCs, the intracellular trafficking and metabolite formation was investigated and KSP inhibition was confirmed as the ADC mode of action. Depending on the linker composition differential profiles of the ADC metabolites with regard to efflux, cellular permeation, and bystander effect have been achieved. Moreover, specific accumulation in the tumor versus other tissues was demonstrated in biodistribution studies in vivo. In conclusion, KSP inhibitors have been established as a versatile new payload class for the generation of highly potent and selective ADCs. Citation Format: Hans-Georg Lerchen, Sven Wittrock, Nils Griebenow, Mario Lobell, Anne-Sophie Rebstock, Yolanda Cancho-Grande, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Anette Sommer, Sandra Berndt, Carsten Terjung, Heiner Apeler, Bertolt Kreft, Rolf Jautelat. Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2017-3234

Abstract 46: Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens

Abstract Antibody-drug conjugates (ADCs) are promising agents that are developed for targeted delivery of cytotoxic payloads to tumor cells. ADCs share a common design of antibody, linker, and cytotoxic payload. Despite significant efforts, the number of available payload classes with a differentiated mode-of-action that can successfully be employed to generate antibody-drug conjugates (ADCs) is still rather limited. So far, only ADCs with microtubule depolymerizing or DNA binding payloads have been approved. The identification of ADC payload classes with a novel mode-of-action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of the kinesin spindle protein (KSP/Eg5/KIF11) have generated interest due to their high anti-tumor activity. However, the transfer of the potency of small molecule KSP inhibitors (KSPis) to highly efficient clinical regimens with a sufficient therapeutic window remains challenging. Through the conjugation of a novel pyrrole subclass of KSPis to antibodies targeting different cancer antigens, we generated a panel of ADCs and characterized them both in vitro and in vivo. ADCs targeting either EGFR or TWEAKR/Fn14 showed strong and specific internalization and displayed specific and potent anti-proliferative efficacy in vitro. In cytotoxicity assays, these ADCs exhibited sub-nanomolar potency in antigen-positive cancer cell lines (EGFR/TWEAKR-pos. NCI-H292; TWEAKR-pos. BxPC3, LoVo) and more than 100-fold selectivity versus non-targeted control-ADC containing the same linker and the same payload. Furthermore, selective anti-tumor efficacy of EGFR- and TWEAKR-KSPi-ADCs was demonstrated in vivo using both cancer cell line-derived models of NSCLC (NCI-H292), urothelial cell carcinoma (UCC) (KU-19-19), and renal cell carcinoma (A498), as well as in the TWEAKR-positive patient-derived xenograft UCC model BFX469. At doses of 5-10 mg/kg qw or bw potent anti-tumor efficacy with treated-to-control ratios (T/C) between 0.16 to 0.28 as well as complete regressions were observed. In summary, KSP inhibitors have been established as a promising new payload class allowing the generation of highly potent and selective ADCs for the treatment of solid tumors. Citation Format: Anette Sommer, Sandra Berndt, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Sven Wittrock, Anne-Sophie Rebstock, Lisa Dietz, Christoph Mahlert, Simone Greven, Nils Griebenow, Yolanda Cancho-Grande, Rolf Jautelat, Heiner Apeler, Bertolt Kreft. Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 46. doi:10.1158/1538-7445.AM2017-46

A better angel of our nature: Hanna Michel "Jean" Khoury, MD (April 24, 1967-May 22, 2017).

A better angel of our nature: Hanna Michel "Jean" Khoury, MD (April 24, 1967-May 22, 2017).

Multicomponent Assembly of the Kinesin Spindle Protein Inhibitor CPUYJ039 and Analogues as Antimitotic Agents.

The potent kinesin spindle protein inhibitor CPUYJ039 and a set of analogues were prepared by a target-oriented approach based on a Ugi reaction that uses 2-nitrophenyl isocyanides as key building blocks. The herein documented strategy provides straightforward and atom economical access to potent benzimidazole-based antimitotic agents by exploring the versatility and exploratory power of the Ugi reaction. The results of docking studies and biological activity evaluations of the benzimidazole compounds are also reported.

Safety and Efficacy of Filanesib in Combination with Pomalidomide and Dexamethasone in Refractory MM Patients. Phase Ib/II Pomdefil Clinical Trial Conducted By the Spanish MM Group

BackgroundFilanesib (ARRY-520) is a kinesin spindle protein inhibitor, with clinical activity in monotherapy in heavily pretreated MM patients (Lonial ASH 2013). We have previously demonstrated the preclinical synergy of this compound with pomalidomide and dexamethasone (Hernandez-García ASH 2015 & EHA 2016), the basis for the present trial.Material and MethodsMM patients relapsing after at least two prior lines of therapy including bortezomib and lenalidomide, who were refractory or intolerant to lenalidomide and refractory to the last line of therapy were treated until progression with 28-day cycles of filanesib iv on days 1, 2, 15 & 16, + pomalidomide po days 1-21 + dexamethasone at a fixed dose of 40 mg po days 1, 8, 15 & 22. Mandatory primary prophylaxis with G-CSF comprised two 7-days courses starting at day +3 and day +17. A Phase Ib was initially conducted to evaluate the maximum tolerated dose (MTD) of the combination, using a modified 3+3 dose-escalating algorithm and once the MTD was determined, the Phase II part of the trial was activated in order to evaluate the activity and further define the toxicity of the combination at this dose.ResultsThe phase Ib part of the trial included 14 patients with a median of 3.5 prior lines of treatment (range: 2-6); 11 (79%) were refractory to bortezomib; 13 (93%) refractory to lenalidomide, nine of them (64%) Len-refractory in the last line of therapy; 10 (71%) were double refractory to PI and lenalidomide; and 13 (93%) had received a prior ASCT.Out of these 14 patients, 7 were included in DL1 (pomalidomide 4 mg and filanesib 1 mg/m2) and the other 7 in DL2 (pomalidomide 4 mg and filanesib 1.25 mg/m2). None of the patients in DL1, experienced DLTs; and there was 1 DLT (G3 diarrhea lasting >7 days) in 1 out of the 6 evaluable patients in DL2.The most frequent treatment-related AEs were hematological: Anemia in 50% patients (36% G3 and 14% G4); neutropenia in 93% (21% G2, 21% G3 and 50% G4); and thrombocytopenia in 78% (50% G1/2, 14% G3 and 14% G4). Non hematological related AEs were mainly low grades, the most frequent-ones being diarrhea (28% G1/2 and 7% G3), asthenia in 36% (all of them G1/2) and infections. Most infections were upper respiratory tract and grades 1/2 (43%). Three patients presented with pneumonia (1 G2 and 2 G3), 1 patient each with sepsis and febrile neutropenia (both G3) and 1 patient died due to an Influenza A pneumonia. In order to maintain a good safety profile, although no formal MTD could be established based on DLTs, DL2 was defined as the recommended dose to proceed to the phase 2.As far as efficacy was concerned, 1 patient (7%) achieved VGPR and 6 (43%) PR for an overall response rate (ORR) of 50%; 1 additional patient (7%) achieved MR and the disease was stabilized in the remaining 6 patients (43%) (lasting for at least 3 cycles) accounting for a disease control rate (DCR) of 100%. Median time to response was 3 months. Remarkably, responses were not different in the nine patients refractory to lenalidomide in the last line of therapy (4 PR (44%) and 1 MR (11%)). After a median follow-up of 7 months, the median PFS was 7 months (CI95 3.3-10.6). Only 3 patients have died due to influenza A (n=1) and progressive disease (n=2).ConclusionThe recommended dose of the combination was defined as pomalidomide 4 mg + filanesib 1.25 mg/m2. The main toxicity was hematological but manageable; and the efficacy, with 50% overall response rate and 100% disease control rate in refractory patients, clinically confirms the synergy observed in the preclinical setting and supports the use of filanesib as a good partner for combination with pomalidomide and dexamethasone. Phase II is ongoing and updated results will be presented at the meeting. DisclosuresMartínez-López:Novartis: Honoraria, Speakers Bureau. De La Rubia:Amgen, Bristol Myers, Celgene, Janssen: Consultancy. Ptaszynski:Array BioPharma: Employment. Tunquist:Array BioPharma: Employment. Oriol:Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees. San-Miguel:BMS: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Amgen: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees; MSD: Membership on an entity’s Board of Directors or advisory committees; Millennium: 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. Mateos:Takeda: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Functional 1,3a,6a-triazapentalene scaffold: Design of fluorescent probes for kinesin spindle protein (KSP)

1,3a,6a-Triazapentalene is a compact fluorescent chromophore. In this study, triazapentalene was used to modify a series of biphenyl-type inhibitors of kinesin spindle protein (KSP) to develop fluorescent probes for the intracellular visualization of this protein. Microscopic studies demonstrated that these novel triazapentalene-labeled compounds exhibited inhibitory activity towards KSP in cultured cells and provided important information concerning the intracellular distribution.

Abstract 4828: Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma cell lines

Abstract As a single agent, melphalan is used as a preparative treatment prior to autologous stem cell transplant (ASCT) in patients with multiple myeloma (MM) given its myelosuppressive and anti-myeloma effects. While most patients achieve complete remission following ASCT, a majority will develop recurrent disease. Therefore, novel combinations that improve the anti-myeloma activity of melphalan may improve post-transplant outcomes. The first-in-class agent Filanesib (Fil) is a kinesin spindle protein inhibitor which induces mitotic arrest/apoptosis in proliferating cells and has demonstrated anti-myeloma activity in vitro and in vivo. In this study, we tested the effects of combination treatment with filanesib and melphalan in the human myeloma cell lines (HMCLs), U266, MM1S and NCI-H929. As single agents, melphalan and filanesib both inhibit myeloma cell proliferation as assessed by the MTS assay. However, when HMCLs were treated simultaneously with both drugs an antagonistic interaction was observed. This antagonism was in part due to cell cycle arrest in the S-phase, which prevented filanesib from downregulating the anti-apoptotic proteins, BCL-2 and MCL-1 and exerting its cytotoxic effect in the G2/M-phase.. To elucidate whether sequential treatment could enhance cytotoxicity, HMCLs were treated with either melphalan (1 hour) followed by filanesib (MelFil) for 48 hours or filanesib for 48 hours followed by 1 hour melphalan treatment (FilMel). Compared to the apoptosis observed with individual drugs, the MelFil sequence led to significantly lower apoptosis, whereas the the FilMel treatment led to enhanced apoptosis following 48 hours after drug washout. Furthermore examination of recovery of cell proliferation following seven days of recovery after drug removal revealed an increase in cell proliferation for the MelFil treatment compared to a decline in the number of viable cells for FilMel treatment. Taken together, our findings suggest that filanesib pretreatment prior to melphalan may be a clinically relevant therapeutic combination strategy. Moreover, our data underscore the importance of testing sequencing strategies for optimizing therapeutic efficacy of combination treatment. Citation Format: Darla Destephanis, Eric J. Norris, Brian Turnquist, Saad Usmani, Mahrukh Ganapathi, Ram Ganapathi. Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4828.

Abstract 3514: Inhibition of PI-3 kinase signaling potentiates the apoptotic response to mitotic arrest

Abstract Anti-mitotic drugs represent a common strategy for cancer chemotherapy by inducing a prolonged prometaphase delay and eventual cell death by apoptosis. Kinesin Spindle Protein (KSP) inhibitors arrest cells in mitosis by blocking bipolar spindle assembly, and these drugs have demonstrated limited side effects in clinical trials. However, they are not as effective as Taxol, whose efficacy is limited by the frequent development of resistance as well as the deleterious effects on non-dividing cells. Recent studies suggest that the ability of cells to maintain energy levels during mitosis could account for the differential responses of cancer cells to mitotic arrest. To determine if manipulating cellular metabolism could potentiate the efficacy of KSP inhibitors, we arrested cells in mitosis in the absence or presence of the PI3-kinase LY294002, and probed for the induction of apoptosis. Because tumor cells respond differently to mitotic arrest, we employed two cell lines that either survive mitotic arrest (MCF7) or undergo apoptosis shortly following mitotic slippage (HeLa). In both cases, simultaneous inhibition of KSP and PI3-kinase induced apoptosis more effectively than mitotic arrest or PI3-K inhibition alone. In the case of HeLa cells, there also appeared to be an acceleration of cell death such that arrested cells underwent apoptosis prior to mitotic slippage. Similar effects were observed using Akt inhibitors, providing additional evidence that PI3-kinase signaling was critical for surviving mitotic arrest. Lastly, we found that increased glucose had a protective effect, further supporting the notion that the ability to survive mitotic arrest depended on the ability to maintain energy levels. If confirmed, this may represent a novel approach for potentiating the anti-tumor activity of KSP inhibitors in the clinic. Citation Format: Roaa M. Kassim, Charles B. Shuster. Inhibition of PI-3 kinase signaling potentiates the apoptotic response to mitotic arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3514.

New investigational drugs with single-agent activity in multiple myeloma.

The treatment of multiple myeloma (MM) is rapidly evolving. In the United States, four drugs (panobinostat, ixazomib, daratumumab and elotuzumab) were approved for the treatment of MM in 2015. As a result of improved diagnosis and therapy, there has been a dramatic improvement in the outcome of MM in the last decade, probably more than any other malignancy. Numerous agents continue to be studied in preclinical models and in clinical trials, with many demonstrating clinical efficacy that appears promising enough to have a trajectory for regulatory approval. The purpose of this article is to summarize the current data and provide perspective on new investigational agents with promising single-agent activity in MM. The agents reviewed include Isatuximab, an anti-CD38 monoclonal antibody; marizomib, a new proteasome inhibitor; oprozomib, an oral proteasome inhibitor; filanesib (ARRY-520), a kinesin spindle protein inhibitor; dinaciclib, a cyclin-dependent kinase inhibitor; venetoclax (ABT-199), a selective BCL-2 inhibitor; and LGH-447, pan PIM kinase inhibitor.

KSP inhibitor SB743921 inhibits growth and induces apoptosis of breast cancer cells by regulating p53, Bcl-2, and DTL.

Kinesin spindle protein (KSP) is a microtubule-associated motor protein that is specifically expressed by mitosis cells. It is highly expressed in various types of tumors including hematomalignances and solid tumors. Chemical KSP inhibition has become a novel strategy in the development of anticancer drugs. SB743921 is a selective inhibitor for KSP, which is a mitotic protein essential for cell-cycle progression. Although SB743921 has shown antitumor activities for several types of cancers and entered into clinical trials, its therapeutic effects on breast cancer and mechanisms have not been explored. In this study, we tested the antitumor activity of SB743921 in breast cancer cell lines and partly elucidated its mechanisms. KSP and denticleless E3 ubiquitin–protein ligase homolog (DTL) are overexpressed in breast cancer cells compared with no-cancer tissues. Chemical inhibition of KSP by SB743921 not only reduces proliferation but also induces cell-cycle arrest and leads to apoptosis in breast cancer cells. Treatment of MCF-7 and MDA-MB-231 breast cancer cell lines with SB743921 results in decreased ability of colony formation in culture. SB743921 treatment also causes a KSP accumulation in protein level that is associated with cell arrest. Furthermore, we showed that SB743921 treatment significantly reduces the expression of bcl-2 and cell cycle-related protein DTL, and upregulates p53 and caspase-3 in breast cancer cells. Taken together, these data indicated that SB743921 can be expected to be a novel treatment agent for breast cancers.

Novel agents in the treatment of multiple myeloma: a review about the future.

Multiple myeloma (MM) is a disease that affects plasma cells and can lead to devastating clinical features such as anemia, lytic bone lesions, hypercalcemia, and renal disease. An enhanced understanding of MM disease mechanisms has led to new more targeted treatments. There is now a plethora of treatments available for MM. In this review article, our aim is to discuss many of the novel agents that are being studied or have recently been approved for the treatment of MM. These agents include the following: immunomodulators (pomalidomide), proteasome inhibitors (carfilzomib, marizomib, ixazomib, oprozomib), alkylating agents (bendamustine), AKT inhibitors (afuresertib), BTK inhibitors (ibrutinib), CDK inhibitors (dinaciclib), histone deacetylase inhibitors (panobinostat, rocilinostat, vorinostat), IL-6 inhibitors (siltuximab), kinesin spindle protein inhibitors (filanesib), monoclonal antibodies (daratumumab, elotuzumab, indatuximab, SAR650984), and phosphoinositide 3-kinase (PI3K) inhibitors.

Treatment of MM: Upcoming Novel Therapies.

Treatment for myeloma has dramatically changed over the past decade, as has overall survival, due in large part to the development of new targeted agents. While proteasome inhibitors and immunomodulatory agents have contributed to improved outcomes, additional new options remain an unmet medical need. Classes of emerging agents include those targeting epigenetics, such as histone deacetylase inhibitors, monoclonal antibodies, and other emerging targets, such as kinesin spindle protein (KSP) inhibitors, cyclin dependent kinase (CDK) inhibitors, and nuclear protein export inhibitors. Future treatment approaches will need to identify how and when to incorporate these treatment options to optimally treat patients with relapsed or refractory myeloma.

Filanesib Primarily Initiates the Apoptotic Program By Activating Bax through a Calpain-Dependent Mechanism

Introduction:Filanesib (ARRY-520) is a highly selective inhibitor of kinesin spindle protein (KSP), a microtubule motor protein active in proliferating cells, which plays an essential role in assembly and maintaining of the bipolar spindle. In cells arrested by KSP inhibition, Mcl-1 is rapidly depleted resulting in cell death, and consequently cells that are dependent on this pro-survival protein, such as myeloma cells, are particularly sensitive to filanesib. We investigated the mechanisms underlying the antimyeloma effect of this agent, focusing on other Bcl-2 family members.Methods: In vitro action of filanesib, alone and in combination with calpain inhibitor PD150606, was evaluated in multiple myeloma (MM) cell lines by MTT assay, Annexin V staining and cell cycle profile analysis by flow cytometry. MM cells were transiently transfected with non- targeting control short interfering RNA (NT-siRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Expression levels of different proteins were analyzed by Western-Blot.Results:We previously showed that all 11 MM cell lines tested were sensitive to filanesib and that sensitivity to this agent correlated with Bax levels. For these experiments, we focused on 3 cell lines with different Bax expression and sensitivity to filanesib: OPM-2 and MM1S (sensitive and high Bax levels) and U266 (less sensitive and low Bax levels). Treatment of MM1S with this agent triggered the translocation to the mitochondria of several proapoptotic Bcl-2 family members such as Noxa, Bim and Bax with several downstream effects. The mitochondrial translocation and activation of Noxa is key in the degradation of Mcl-1 by mediating the translocation of this protein from the cytosol to the mitochondria promoting its degradation. Regarding Bim, filanesib also induced the early (12-24 hours) expression of several proapoptotic isoforms of Bim that also translocated to the mytochondria. As previously reported, Bax is the top determinant of sensitivity to filanesib. In the present study, remarkably, once translocated into the mitochondria, Bax was also cleaved into the very potent proapoptotic 18 kDa fragment. All these events triggered the mitochondrial release of cytochrome C (caspase dependent apoptosis) and AIF (caspase independent apoptosis).In order to confirm the role of Bax in filanesib-induced apoptosis, we knocked-down Bax in MM1S by using small interfering RNA. This approach clearly decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced only 26% apoptosis in the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib).Furthermore, treatment with filanesib also induced cleavage of effector caspases (3 and 7) in all cell lines studied (OPM2, MM1S, U266). PARP was also cleaved in these cells, but it was previous to caspase activation in the most sensitive cell lines (OPM2, MM1S) suggesting a caspase-independent mechanism of apoptosis. This was confirmed by pre-treatment with the pan caspase-inhibitor Z-VAD-FMK, which did not rescue OPM2 and MM1S cells from apoptosis.Interestingly, one potential mechanism that could link both effects is the activity of calpain, a cysteine protease involved in caspase-independent apoptosis. This protein is a well-known caspase-independent way of processing PARP into the 60 kDa fragment, and has also been described as being responsible for Bax cleavage into the 18-kDa fragment. Consistent with this hypothesis, pre-treatment with the calpain inhibitor PD150606 clearly reduced the activity of filanesib in these cells (35 % to 70 % of survival) as assessed by MTT.Finally, consistent with the previous hypothesis, the less sensitive U266 cell line contained undetectable Bax protein suggesting that filanesib was not able to trigger caspase-independent apoptosis. However, a secondary caspase dependent apoptosis mechanism was confirmed as the pan-caspase inhibitor ZVAD-FMK was able to almost completely abrogate the activity of filanesib.Conclusions: Our results show that filanesib primarily initiates apoptosis by activating Bax in a caspase-independent manner, probably via calpain, a powerful accelerator of the apoptotic process. In addition, Noxa and BIM appear to be crucial for modulating Mcl-1 proteasomal degradation and Bax activation.This work was funded in part by the company Array BioPharma. DisclosuresTunquist:Array BioPharma: Employment. Mateos:Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Ocio:Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding; MSD: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Pharmamar: Consultancy, Research Funding; Janssen: Honoraria.

Mimicking the Myeloma Niche: A 3D Bone-Derived Co-Culture System to Selectively Assess Bystander BMSCs and to Perform High-Throughput Drug Screening in Multiple Myeloma

Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM.Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances.Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e).Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. [Display omitted] DisclosuresEngelhardt:Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.