Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of ...immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.
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
and
models. Mechanisms of this synergistic combination were dissected by gene expression profiling, immunostaining, cell cycle and short interfering ribonucleic acid studies. Filanesib showed
,
, and
synergy with pomalidomide plus dexamethasone treatment. Importantly, the
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.
Despite recent advances in the treatment of multiple myeloma (MM), the prognosis of most patients remains poor, and resistance to traditional and new drugs frequently occurs. EDO-S101 is a novel ...therapeutic agent conceived as the fusion of a histone deacetylase inhibitor radical to bendamustine, with the aim of potentiating its alkylating activity.
The efficacy of EDO-S101 was evaluated in vitro, ex vivo and in vivo, alone, and in combination with standard anti-myeloma agents. The underlying mechanisms of action were also evaluated on MM cell lines, patient samples, and different murine models.
EDO-S101 displayed potent activity in vitro in MM cell lines (IC
1.6-4.8 μM) and ex vivo in cells isolated from MM patients, which was higher than that of bendamustine and independent of the p53 status and previous melphalan resistance. This activity was confirmed in vivo, in a CB17-SCID murine plasmacytoma model and in de novo Vk*MYC mice, leading to a significant survival improvement in both models. In addition, EDO-S101 was the only drug with single-agent activity in the multidrug resistant Vk12653 murine model. Attending to its mechanism of action, the molecule showed both, a HDACi effect (demonstrated by α-tubulin and histone hyperacetylation) and a DNA-damaging effect (shown by an increase in γH2AX); the latter being again clearly more potent than that of bendamustine. Using a reporter plasmid integrated into the genome of some MM cell lines, we demonstrate that, apart from inducing a potent DNA damage, EDO-S101 specifically inhibited the double strand break repair by the homologous recombination pathway. Moreover, EDO-S101 treatment reduced the recruitment of repair proteins such as RAD51 to DNA-damage sites identified as γH2AX foci. Finally, EDO-S101 preclinically synergized with bortezomib, both in vitro and in vivo.
These findings provide rationale for the clinical investigation of EDO-S101 in MM, either as a single agent or in combination with other anti-MM drugs, particularly proteasome inhibitors.
Background: Venetoclax is a BCL-2 inhibitor particularly effective in patients with multiple myeloma (MM) harboring the t(11;14). However, resistance to venetoclax has been linked to MCL-1 ...overexpression. On the other hand, it is wellknown that MM cells depend on MCL-1 rather than BCL-2 for survival, and this dependence has recently been reported to be enhanced by the tumor-associated microenvironment. Therefore, the combination of venetoclax with the potent MCL-1 inhibitor S63845 arises as a promising and novel approach for the treatment of MM.
Aims: To evaluate the efficacy and mechanism of action of S63845 alone and in combination with venetoclax in absence and presence of the bone marrow tumor microenvironment in preclinical in vitro, ex vivo and in vivo models of MM.
Methods: S63845 was provided by an agreement with Servier and Novartis. In vitro activity of S63845 and venetoclax alone and in combination was evaluated by bioluminescence on a MM cell line expressing luciferase (MM.1S-luc) in absence and presence of mesenchymal stromal cells isolated from bone marrow aspirates of MM patients (pMSCs). MM.1S cells cultured in absence or presence of pMSCs were analyzed for MCL-1 and BCL-2 protein levels by Western blot. Interactions between these anti-apoptotic proteins with the pro-apoptotic protein BIM were assessed by immunoprecipitation assays. The efficacy of S63845 and venetoclax alone and in combination was also evaluated ex vivo in MM cells and normal lymphocytes from MM patients. Finally, a disseminated MM model in BRG mice was used for in vivo studies.
Results: S63845 and venetoclax showed a strong antimyeloma dose-dependent effect on MM.1S-luc cells co-cultured with pMSCs. However, whereas the presence of tumor-associated MSCs increased the IC50 value of venetoclax in MM.1S-luc cells from 6.2 to 9.8 mM, it reduced that of S63845 from 94.1 to 81 nM, suggesting a mild sensitization to this drug in the context of the microenvironment. Neither S63845 nor venetoclax affected pMSC viability even at high concentrations by MTT assay. The co-culture with the BM stromal microenvironment increased MCL-1 expression on untreated MM.1S cells in two out of four experiments performed with MSCs from different MM patients, whereas it surprisingly induced a decrease on BCL-2 levels in all of them. Treatment with S63845 completely blocked MCL-1 binding to BIM, both in the absence or presence of pMSCs but did not induce the compensatory increase of BCL-2/BIM complexes observed in MM.1S cells in monoculture. Venetoclax also completely blocked the binding of BCL-2 to BIM in MM.1S alone or in co-culture, and induced a similar compensatory increase of MCL-1/BIM complexes in both situations. Importantly, the double combination S63845 + venetoclax was significantly superior to both drugs in monotherapy in killing MM.1S-luc cells co-cultured in the presence of the stromal microenvironment. BIM immunoprecipitation assays showed that the double combination was able to counteract the compensatory upregulation of MCL-1 bound to BIM observed on MM.1S cells treated with venetoclax and to entirely disrupt BCL-2/BIM complexes, both in the absence and presence of pMSCs. Furthermore, S63845 + venetoclax increased the percentage of apoptotic MM plasma cells from three MM patients with respect to single treatments with moderate toxicity detected on normal lymphocytes, suggesting the existence of a therapeutic window for the double combination. Finally, the combination of S63845 + venetoclax clearly delayed tumor growth as compared with the agents in monotherapy in a disseminated model of MM with statistically significant differences from day 19 of treatment. This in vivo effect translated into a significatively improved survival for mice treated with the double combination (median 60 days) vs control mice (median 32 days; log-rank test P=0.045).
Conclusion: Our preclinical data demonstrate the potent activity of the combination of venetoclax with S63845 in MM even in presence of the stromal associated-tumor microenvironment, and provides the rationale for the clinical development of this combination in relapsed or refractory MM patients.
This project was supported by Novartis Pharmaceuticals and by the Spanish , ISCIII-FIS PI15/00067 and PI15/02156, GRS 1604/A/17 and CRMRTC de Castilla y León. Predoctoral grant to EMA by Consejería de Educación de Castilla y León.
Schoumacher:Servier: Employment. Banquet:Servier: Employment. Kraus-Berthier:servier: Employment. Kloos:Servier: Employment; Novartis: Other: Partnership. Halilovic:Novartis: Employment, Equity Ownership. Maacke:Novartis: Employment. Mateos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ocio:AbbVie: Consultancy; Novartis: Consultancy, Honoraria; BMS: Consultancy; Seattle Genetics: Consultancy; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Pharmamar: Consultancy; Sanofi: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding.
Background: Proviral Insertion site for Moloney murine leukemia virus (PIM) kinases are overexpressed in hematologic malignancies, including multiple myeloma. Previous preclinical data from our group ...demonstrated the anti-myeloma effect of the pan-PIM kinase inhibitor PIM447. Methods: Based on those data, we evaluate here, by in vitro and in vivo studies, the activity of the triple combination of PIM447 + pomalidomide + dexamethasone (PIM-Pd) in multiple myeloma. Results: Our results show that the PIM-Pd combination exerts a potent anti-myeloma effect in vitro and in vivo, where it markedly delays tumor growth and prolongs survival of treated mice. Mechanism of action studies performed in vitro and on mice tumor samples suggest that the combination PIM-Pd inhibits protein translation processes through the convergent inhibition of c-Myc and mTORC1, which subsequently disrupts the function of eIF4E. Interestingly the MM pro-survival factor IRF4 is also downregulated after PIM-Pd treatment. As a whole, all these molecular changes would promote cell cycle arrest and deregulation of metabolic pathways, including glycolysis and lipid biosynthesis, leading to inhibition of myeloma cell proliferation. Conclusions: Altogether, our data support the clinical evaluation of the triple combination PIM-Pd for the treatment of patients with multiple myeloma.
BH3-mimetics targeting anti-apoptotic proteins such as MCL-1 (S63845) or BCL-2 (venetoclax) are currently being evaluated as effective therapies for the treatment of multiple myeloma (MM). ...Interleukin 6, produced by mesenchymal stromal cells (MSCs), has been shown to modify the expression of anti-apoptotic proteins and their interaction with the pro-apoptotic BIM protein in MM cells. In this study, we assess the efficacy of S63845 and venetoclax in MM cells in direct co-culture with MSCs derived from MM patients (pMSCs) to identify additional mechanisms involved in the stroma-induced resistance to these agents. MicroRNAs miR-193b-3p and miR-21-5p emerged among the top deregulated miRNAs in myeloma cells when directly co-cultured with pMSCs, and we show their contribution to changes in MCL-1 and BCL-2 protein expression and in the activity of S63845 and venetoclax. Additionally, direct contact with pMSCs under S63845 and/or venetoclax treatment modifies myeloma cell dependence on different BCL-2 family anti-apoptotic proteins in relation to BIM, making myeloma cells more dependent on the non-targeted anti-apoptotic protein or BCL-X
. Finally, we show a potent effect of the combination of S63845 and venetoclax even in the presence of pMSCs, which supports this combinatorial approach for the treatment of MM.
Despite new advances in multiple myeloma treatment and the consequent improvement in overall survival, most patients relapse or become refractory to treatment. This suggests that new molecules and ...combinations that may further inhibit important survival pathways for these tumor cells are needed. In this context, zalypsis is a novel compound, derived from marine organisms, with a powerful preclinical anti-myeloma effect based on the sensitivity of malignant plasma cells to DNA-damage induction; and it has already been tested in a phase I/II clinical trial in multiple myeloma. We hypothesized that the addition of this compound to the combination of bortezomib plus dexamethasone may improve efficacy with acceptable toxicity. The triple combination demonstrated strong synergy and higher efficacy compared with double combinations; not only in vitro, but also ex vivo and, especially, in in vivo experiments. The triple combination triggers cell death, mainly through a synergistic induction of DNA damage and a decrease in the nuclear localization of nuclear factor kappa B. Our findings support the clinical evaluation of this combination for relapsed and refractory myeloma patients.
Endoscopic ultrasound-guided liver biopsy (EUS-LB) has been proposed as a novel technique that could offer some advantages over traditional methods, especially regarding specimen adequacy. A ...systematic review that included 32 studies evaluating the quality of percutaneous hepatic biopsies (PC-LB) demonstrated that the average number of portal tracts with this technique was 7.5 +/-3.4. (1) The objective of our study was to determine if EUS-LBs meet AASLD quality criteria, defined by the presence of more than 11 portal tracts.
A retrospective study was carried out from a prospectively created EUS-LB database. The primary objective was to evaluate the sample quality, using as a parameter the number of portal tracts. The secondary objective was to determine the security profile of the procedure and evaluate the rate of complications.
82 patients were included (average age 55). The main indication for tissue acquisition was elevated transaminases. Steatosis/steatohepatitis was the most common histological diagnosis. The average number of portal tracts was 19.23 +/- 7.2. All the samples had at least 11 portal tracts. The rate of adverse events was 9.75%. The majority were minor complications (post-procedure pain). Only one patient presented a severe complication, bleeding secondary to an arterio-biliary fistula, that required embolization by interventional radiology.
EUS-LBs meet quality criteria established by AASLD, have an excellent security profile, and might be considered the method of choice for liver tissue acquisition in the centers where the resource is available.
Abstract 134
The introduction of novel agents has improved the outcome of MM patients, but MM is still considered an incurable disease and the emergence of resistance is the main responsible for this ...situation. The unraveling of the resistance mechanisms would help to design novel therapeutic strategies (combinations or sequencing treatments) to overcome this problem.
We have developed an in vivo model of acquired resistance to antimyeloma agents based in a model of subcutaneous plasmocytoma (MM1S) in CB17-SCID mice. For this purpose, mice were treated with Lenalidomide (25 mg/Kg) + Dex (1 mg/Kg) (LD), Pomalidomide (5 mg/Kg) + Dex (PD) or vehicle control, and after a period of initial sensitivity, tumors developed resistance to the administered combination. At this moment selected sensitive and resistant tumors were excised to analyze apoptosis, signaling pathways and gene expression profiling (GEP) changes. Moreover, some of these mice bearing resistant tumors were switched to receive the alternative combination (that is LD-PD or PD- LD), and, in selected mice, a second change of treatment was performed after secondary resistance to receive again the initial treatment (LD-PD-LD or PD-LD-PD). In order to evaluate TTP and to define the moment to change treatment we considered progression when tumor volume reached 1.700 mm3.
Both combinations (LD & PD) controlled the initial growth of the tumors, with a higher potency for the PD combination. Tumor volume reached 500 mm3 at a median of 8, 42 & 53 days for control, LD and PD respectively (p=0.01 and p=0.001). Nevertheless, after 30 days of continuous treatment, and despite maintaining the administration of the drugs, tumors started growing, and, once the tumors had reached 500 mm3, their growth kinetic was similar for the treated mice (despite still being treated) as compared to the untreated mice, indicating the emergence of complete resistance. This resistance was also confirmed ex vivo by in vitro culture with the corresponding drugs.
In order to test the presence of cross-resistance, mice bearing big tumors resistant to LD or PD and already growing in an exponential phase (volume of 1.700 mm3), were at this point treated with the alternative combination. This sequential treatment change induced tumor stabilization and even a decrease of tumor volume. Again PD was significantly more potent at overcoming LD resistance as compared to the alternative situation, and this was verified both in terms of tumor growth inhibition (p=0.005) and in terms of time to progression (median of 16 vs 27 days for LD and PD respectively. p=0.004).
Furthermore, mice that had been treated with LD-PD or PD-LD and had developed resistance to both combinations were again treated with the initial combination, and, surprisingly, they were again sensitive, indicating the reversibility of the acquired resistance. Similarly to previous experiments, PD was again significantly more potent than LD. This reversibility was also confirmed ex vivo after culture of the cells in medium without drugs.
In order to investigate the resistance mechanisms, cells extracted from sensitive and resistant tumors were analyzed by Western Blot. Treatment with LD and PD induced a downregulation of pERK 1/2, nevertheless, when these cells developed resistance a very significant increase of pERK 1/2 levels (even higher than the basal levels) was observed. Moreover, these resistant cells also showed an upregulation of p-MEK, p-RAF and RAS. In this same line, the MEK inhibitor PD-98059 potentiated the in vitro activity of LD or PD in fresh MM1S cells, with high synergism (CI<0.1).
Finally, changes in GEP were evaluated in extracted tumors. A significant change was observed in cells from tumors sensitive to PD and LD (with a higher gene deregulation with the former combination), as compared to untreated ones. Interestingly, when tumors became resistant, most of these changes disappeared and the GEP partially returned to a profile similar to that of active untreated tumors.
The data presented would support treatment with alternative IMIDs if resistance was developed to one of them (specially PD was significantly more potent at rescuing resistance to LD), or even the retreatment with the same IMID after a wash up period. Moreover this study supports the evaluation of combinations of IMIDs with agents that abrogate the ERK pathway in order to increase efficacy or avoid resistance.
Quintana:Celgene: Employment. García:Celgene: Employment. Pandiella:Celgene: Research Funding. San Miguel:Celgene: Consultancy, Research Funding.
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