scholarly journals SUMOylation inhibition enhances dexamethasone sensitivity in multiple myeloma

2022 ◽  
Vol 41 (1) ◽  
Author(s):  
Li Du ◽  
Wei Liu ◽  
Grace Aldana-Masangkay ◽  
Alex Pozhitkov ◽  
Flavia Pichiorri ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Models ◽  
Cell Lines ◽  
Single Agent ◽  
Significant Negative Correlation ◽  
Therapeutic Drug ◽  
Rna Seq ◽  
Key Factors ◽  
Microrna Sequencing

Abstract Background Multiple myeloma (MM) is an incurable plasma cell malignancy. Although Dexamethasone (Dex) is the most widely used therapeutic drug in MM treatment, patients develop Dex resistance leading to progressive disease, demanding an urgent need to investigate the mechanisms driving Dex resistance and develop new reagents to address this problem. We propose SUMOylation as a potential mechanism regulating Dex resistance and SUMOylation inhibition can enhance Dex sensitivity in MM. Methods Using MM cell lines and primary MM samples from relapsing MM patients, we evaluated the effects of knockdown of SUMO E1 (SAE2) or using TAK-981, a novel and specific SUMO E1 inhibitor, on Dex sensitivity. Xenograft mouse models were generated to determine the in vivo anti-MM effects of TAK-981 as a single agent and in combination with Dex. miRNA-seq, RNA-seq and GSEA analysis were utilized for evaluating key factors mediating Dex resistance. Chromatin immunoprecipitation (ChIP) assay was performed to determine the binding occupancy of c-Myc on promoter region of miRs. Results We observed a significant negative correlation between SUMO E1 (SAE2) expression and Dex sensitivity in primary MM samples. Knockdown of SAE2 or using TAK-981 significantly enhances myeloma sensitivity to Dex in MM cell lines. Moreover, the enhanced anti-MM activity by TAK-981 and Dex combination has been validated using primary relapsing MM patient samples and xenograft mouse models. SUMOylation inhibition increased glucocorticoid receptor (GR) expression via downregulation miR-130b. Using RNA and microRNA sequencing, we identified miR-551b and miR-25 as important miRs mediating Dex resistance in MM. Overexpression of miR-551b and miR-25 caused resistance to Dex, however, knockdown of miR-551b and miR-25 significantly enhanced Dex sensitivity in MM. SAE2 knockdown or TAK-981 treatment downregulated the expression of miR-551b and miR-25, leading to induction of miR targets ZFP36, ULK1 and p27, resulting in apoptosis and autophagy. We demonstrated c-Myc as a major transcriptional activator of miR-130b, miR-551b and miR-25 and SUMOylation inhibition downregulates these miRs level by decreasing c-Myc level. Conclusion Our study proves SUMOylation plays a crucial role in Dex resistance in MM and SUMOylation inhibition appears to be an attractive strategy to advance to the clinic for MM patients.

scholarly journals SUMOylation Inhibition Enhances Dexamethasone Sensitivity in Multiple Myeloma

2021 ◽  
Author(s):  
Li Du ◽  
Wei Liu ◽  
Grace Aldana-Masangkay ◽  
Alex Pozhitkov ◽  
Flavia Pichiorri ◽  
...  
Keyword(s):  
Mouse Models ◽  
Cell Lines ◽  
Chromatin Immunoprecipitation ◽  
Single Agent ◽  
Significant Negative Correlation ◽  
Therapeutic Drug ◽  
Rna Seq ◽  
Key Factors ◽  
Microrna Sequencing

Abstract BackgroundMultiple myeloma (MM) is an incurable plasma cell malignancy. Although Dexamethasone (Dex) is the most widely used therapeutic drug in MM treatment, patients develop Dex resistance leading to progressive disease, demanding an urgent need to investigate the mechanisms driving Dex resistance and develop new reagents to address this problem. We propose SUMOylation as a potential mechanism regulating Dex resistance and SUMOylation inhibition can enhance Dex sensitivity in MM.MethodsUsing MM cell lines and primary MM samples from relapsing MM patients, we evaluated the effects of knockdown of SUMO E1 (SAE2) or using TAK-981, a novel and specific SUMO E1 inhibitor, on Dex sensitivity. Xenograft mouse models were generated to determine the in vivo anti-MM effects of TAK-981 as a single agent and in combination with Dex. miRNA-seq, RNA-seq and GSEA analysis were utilized for evaluating key factors mediating Dex resistance. Chromatin immunoprecipitation (ChIP) assay was performed to determine the binding occupancy of c-Myc on promoter region of miRs. ResultsWe observed a significant negative correlation between SUMO E1 (SAE2) expression and Dex sensitivity in primary MM samples. Knockdown of SAE2 or using TAK-981 significantly enhances myeloma sensitivity to Dex in MM cell lines. Moreover, the enhanced anti-MM activity by TAK-981 and Dex combination has been validated using primary relapsing MM patient samples and xenograft mouse models. SUMOylation inhibition increased glucocorticoid receptor (GR) expression via downregulation miR-130b. Using RNA and microRNA sequencing, we identified miR-551b and miR-25 as important miRs mediating Dex resistance in MM. Overexpression of miR-551b and miR-25 caused resistance to Dex, however, knockdown of miR-551b and miR-25 significantly enhanced Dex sensitivity in MM. SAE2 knockdown or TAK-981 treatment downregulated the expression of miR-551b and miR-25, leading to induction of miR targets ZFP36, ULK1 and p27, resulting in apoptosis and autophagy. We demonstrated c-Myc as a major transcriptional activator of miR-130b, miR-551b and miR-25 and SUMOylation inhibition downregulates these miRs level by decreasing c-Myc level. ConclusionOur study proves SUMOylation plays a crucial role in Dex resistance in MM and SUMOylation inhibition appears to be an attractive strategy to advance to the clinic for MM patients.

scholarly journals A Novel Oncolytic Reovirus Immune Priming Strategy Dramatically Enhances the Efficacy of Anti-PD-L1 Antibody Therapy Against Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3960-3960 ◽  
Author(s):  
Kevin R. Kelly ◽  
Claudia M. Espitia ◽  
Weiguo Zhao ◽  
Valeria Visconte ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Study ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Single Agent ◽  
Bone Destruction ◽  
The United States ◽  
Significant Activity ◽  
Immune Priming

Abstract Multiple myeloma (MM) is the second most common adult hematologic malignancy in the United States and is characterized by a unique form of progressive bone destruction. Despite the development of new treatments such as proteasome inhibitors and immunomodulatory agents, patients with high-risk disease have a median survival of only 2-3 years, highlighting the demand for more effective therapeutic strategies. Oncolytic viral formulations represent a promising new class of anticancer agents that may have important applications in precision medicine. Recent studies have demonstrated that reoviruses specifically replicate only in cancer cells and this led to the development of a reovirus-based oncolytic viral therapy called Reolysin. We have demonstrated that reoviruses selectively replicate in MM cells and Reolysin possesses significant activity in preclinical in vitro and in vivo MM models. These findings established the framework for an ongoing investigator-initiated phase 1b clinical study of Reolysin in combination with bortezomib and dexamethasone in patients with relapsed/refractory MM. Recent gene ontology analyses of RPMI-8226 and U266 MM cells treated with Reolysin revealed that reovirus exposure triggers a highly significant transient increase in CD274(programmed death 1 ligand, PD-L1) in MM cell lines. Reolysin-mediated PD-L1 upregulation was confirmed by immunoblotting, qRT-PCR, and flow cytometric analyses in MM cell lines and primary patient specimens treated with Reolysin. Increased PD-L1 expression was also detected by immunohistochemistry in MM tumor samples collected from mice treated with Reolysin. Adaptive resistance mediated by inhibitory ligands such as PD-L1 has emerged as an important mechanism of malignant cell survival and has led to the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. These agents have exhibited dramatic efficacy in certain forms of cancer including melanoma and lung cancer. Analysis of specimens from patients treated on clinical trials with these agents indicates that high basal expression of PD-L1 on tumor cells may be necessary to elicit significant clinical benefit. Notably, most MM cell lines and primary CD138+ cells from MM patients do not overexpress PD-L1 compared to normal plasma cells and this may preclude patients with MM from optimally benefiting from immune checkpoint therapy. However, novel immune priming strategies that stimulate transient upregulation of PD-L1 on malignant cells could potentially render agents that target the PD-L1/PD-1 axis significantly more effective for a broader range of malignancies including MM. We hypothesized that Reolysin could be used as a precision immune priming agent to potentiate the anti-MM efficacy of PD-L1 targeted therapy by rendering MM cells vulnerable to PD-L1 inhibition through the transient upregulation of target expression. To investigate this therapeutic approach, 5TGM1-luc murine MM cells were injected IV into immunocompetent mice to generate MM bone disease. After disease was established, mice were randomized into groups and treated with vehicle, Reolysin (5 x 108 TCID50, Q7D), murine anti-PD-L1 antibody (200 mg/mouse, Q2D) or the combination for 5 weeks. Mice treated with the combination demonstrated decreased disease burden as measured by bioluminescent imaging and also showed reduced IgG2bk levels (specific IgG secreted by 5TGM1 cells) by ELISA. Importantly, the combination also led to increased overall animal survival compared to vehicle control and either single agent treatment (P<0.01). Analysis of bone marrow specimens from mice in all experimental groups showed that Reolysin stimulated elevated PD-L1 levels in vivo in a manner that was directly linked to the enhanced efficacy of the combination. Our findings demonstrate that Reolysin has dramatic PD-L1-related immune priming effects in clinically relevant models of MM and support its use as a precision agent to sensitize MM cells to immune checkpoint therapy. Based on these promising data, we are currently planning a clinical study of Reolysin in combination with bortezomib and a PD-1 inhibitor in patients with relapsed/refractory MM. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coffey:Oncolytics Biotech: Employment.

Efficacy of Panobinostat (LBH589) in Multiple Myeloma Cell Lines and In Vivo Mouse Model: Tumor-Specific Cytotoxicity and Protection of Bone Integrity in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1510-1510 ◽  
Author(s):  
Joseph D. Growney ◽  
Peter Atadja ◽  
Wenlin Shao ◽  
Youzhen Wang ◽  
Minying Pu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Bone Density ◽  
Mouse Model ◽  
Cortical Bone ◽  
Cell Lines ◽  
Single Agent ◽  
Synergistic Cytotoxicity

Abstract Panobinostat (LBH589) is a highly potent oral pan-deacetylase (DAC) inhibitor currently undergoing clinical development in hematologic and solid malignancies. Here we report the effects of panobinostat on multiple myeloma (MM) cells in vitro and in a murine xenograft model in vivo. Panobinostat exhibited potent cytotoxic activity (IC50 &lt;10 nM) against 8 MM cell lines (KMS-12PE, KMS-18, LP-1, NCI H929, KMS-11, RPMI8226, OPM-2, and U266). Panobinostat has been shown to affect signals involved in MM cell-cycle arrest and cell death, and to induce apoptosis via mitochondrial perturbation. In addition, panobinostat has been shown to selectively induce cell death of plasma cells isolated from MM patients without toxicity to normal lymphocytes or granulocytes. To investigate the effect of panobinostat in vivo, a disseminated luciferized MM.1S xenograft mouse model was treated with vehicle or panobinostat 15 mg/kg by intraperitoneal (i.p.) administration qd×5 for 3 weeks. Panobinostat treatment reduced the burden of MM.1S tumor cells to 22% treated over control (T/C) relative to vehicle-treated animals. In addition, MM.1S tumor-bearing mice treated with panobinostat displayed reduced trabecular and cortical bone damage relative to vehicle-treated animals. The mean ± SEM trabecular bone density and cortical bone density (% Bone Volume/Total Volume) of panobinostat-treated animals was 14.5% ± 2.0 and 98.1% ± 0.4, respectively, compared with 2.2% ± 0.3 and 89.1% ± 1.5 in vehicle-treated animals. In combination with the proteosome inhibitor bortezomib (BZ), panobinostat displayed significant synergistic cytotoxicity without additional toxicity to normal bone marrow stromal cells in vitro. In the MM.1S-luciferase tumor mouse model, combined treatment with panobinostat at 10 mg/kg i.p. qd×5 for 4 weeks and BZ at 0.2 mg/kg intravenously 1qw for 4 weeks reduced tumor burden to 7% T/C relative to vehicle, panobinostat alone (31% T/C), or BZ alone (44% T/C). Disease progression, measured as median time to endpoint (TTE) was improved from 37 to 54 days (P&lt;0.05) by panobinostat and to 46 days by BZ (P&lt;0.05). The combination treatment further improved clinical outcome relative to both single-agent treatment groups (P&lt;0.05), extending the TTE to 73 days. In contrast to BZ, the immunomodulatory drug thalidomide (TH) had no significant single-agent activity at 150 mg/kg p.o. qd for 4 weeks. However, combination activity (18% T/C) was observed when TH was combined with a sub-efficacious dose of panobinostat (5 mg/kg, 64% T/C). Combination of panobinostat and TH increased the TTE to 50 days, compared with 37.5, 43, and 39.5 days (P&lt;0.05), respectively, for the vehicle, panobinostat, or TH as single agents. These data demonstrate that panobinostat exhibits significant anti-proliferative and anti-tumor activities on MM cells both in vitro and in vivo. Panobinostat, as a single agent or in combination with BZ or TH, is a promising therapy for MM, and these studies may provide the rationale for clinical evaluation of panobinostat and BZ combination in the treatment of MM.

scholarly journals Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1945-1945
Author(s):  
Kylee H Maclachlan ◽  
Andrew Cuddihy ◽  
Nadine Hein ◽  
Carleen Cullinane ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Cellular Response ◽  
Single Agent ◽  
Resistant Cell ◽  
Rdna Transcription

Abstract Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity. The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response. Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment. We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials. Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma. References Drygin et al., Cancer Research 2011 Bywater et al., Cancer Cell 2012 Quin et al, Oncotarget, 2016 Devlin et al., Cancer Discovery 2016 Hein et al., Blood 2017 Disclosures Harrison: Janssen-Cilag: Other: Scientific advisory board.

Synergistic Activity of the Proteasome Inhibitor PS-341 with Non-Myeloablative 153-Sm-EDTMP in a Syngeneic, Orthotopic Model of Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3453-3453
Author(s):  
Apollina Goel ◽  
Angela Dispenzieri ◽  
Susan M. Geyer ◽  
Suzanne Greiner ◽  
Stephen J. Russell
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Single Agent ◽  
Myeloma Cell ◽  
Synergistic Activity ◽  
Targeted Radiotherapy ◽  
Myeloma Cells ◽  
Cell Counts

Abstract Multiple myeloma is a highly radiosensitive malignancy but, at the present time, radionuclide-based interventions have no proven place in disease management. Bone-seeking radionuclides such as 153-Sm-EDTMP and 166-Ho-DOTMP are promising agents for systemic delivery of ionizing radiation to sites of myeloma disease activity, but they are associated with significant myelosuppression at therapeutically effective doses and have therefore been used only in the context of myeloma stem cell transplantation protocols. We previously reported that the proteasome inhibitor PS-341 potently and selectively sensitizes myeloma cell lines and primary myeloma cells to the lethal effects of ionizing X-irradiation (Goel et al, Exp Hematol. 33, 784, 2005). To determine whether PS-341 is equally effective in sensitizing myeloma cells to ionizing beta-radiation and to extend our initial observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays were performed using a panel of myeloma cell lines and demonstrated synergistic killing following co-treatment with PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, two doses of PS-341 (0.5 mg/kg), or a single non-myeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22 and 28 days, respectively. In contrast, mice treated with combination therapy comprising two doses of PS-341 (0.5 mg/kg), one day prior to and one day following 153-Sm-EDTMP (22.5 MBq) showed a greatly prolonged median survival of 49 days. Correlative studies indicated that, compared to single-agent therapy, combination treatment with PS-341 and 153-Sm-EDTMP rapidly reduced the clonogenicity of bone-marrow resident 5TGM1 cells, slowed the elevation of serum myeloma-associated paraprotein levels, and was associated with longer term preservation of bone mineral density. The myelotoxicity of single agent and combination therapies was evaluated by comparing peripheral blood cell counts in each of the treatment groups, and by clonogenicity assays of hematopoietic progenitors isolated form normal mice receiving identical treatment regimens. Treatment with 153-Sm-EDTMP led to significant, but transient, myelosuppression which did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. In summary, PS-341 is a potent in vivo radiosensitizer that greatly enhances the therapeutic potency, without increasing myelotoxicity, of skeletal targeted radiotherapy in the syngeneic, orthotopic 5TGM1 myeloma model. Based on these findings, we propose to conduct a phase I clinical trial at Mayo Clinic to evaluate the combination of PS-341 plus non-myeloablative skeletal targeted radiotherapy (using 153-Sm-EDTMP) in patients with advanced or treatment-refractory multiple myeloma.

Dual Inhibition of MDM2 and BET Cooperate to Eradicate Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 674-674 ◽  
Author(s):  
Anne-Louise Latif ◽  
John J Cole ◽  
Joana Monteiro Campos ◽  
William Clark ◽  
Lynn McGarry ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Single Agent ◽  
Rna Seq ◽  
Wild Type ◽  
Advisory Committees ◽  
Bet Inhibitor ◽  
Dual Inhibition

Abstract Background: There remains a critical requirement for novel therapies for Acute Myeloid Leukemia (AML). Bromodomain and extra-terminal domain (BET) inhibitors are emerging as exciting therapeutic agents for hematopoietic malignancies. Pharmacological inhibition of BET bromodomains targets malignant cells by preventing reading of acetylated lysine residues, thus disrupting chromatin-mediated signal transduction, which reduces transcription at oncogenic loci. Although a heterogeneous disease, most AML retains wild type p53. However, p53 is often rendered functionally deficient by over-expression of MDM2. Potentiating the p53 response though MDM2 antagonism is therefore potentially beneficial to most AML subtypes. We hypothesized that dual inhibition of MDM2 and BET would be synthetic lethal to p53 wild type AML. Methods: For in vitro experiments CPI203 (BET inhibitor, Constellation Pharmaceuticals) and nutlin-3 (MDM2 antagonist, Sigma) were assessed on p53 wild type cell lines (OCI-AML3, MOLM-13 and MV411) and p53 wild type primary murine AML. To assess the combination's dependency on wild type p53; p53 mutated cell lines (KG1a, KASUMI-1 and THP1) were tested. Cell viability was assessed using resazurin (Alamar blue dye) across numerous dose ratios on the OCI-AML3 cell line and analysed using the Envision Fluorescent Reader. Drug combination indices (CI) were evaluated using Calcusyn (version 2.0). Apoptosis was assessed using flow cytometry staining for Annexin V and propidium iodide (PI) on all p53 wild type and mutated cell lines. For in vivo experiments CPI0610 (clinical grade BET inhibitor, Constellation Pharmaceuticals) and RG7112 (MDM2 inhibitor, Roche) were tested as single agents, in combination and with relevant vehicle controls. RNA seq was performed on the GAIIX sequencer and gene ontology analysis was performed using DAVID/INGENUITY pathway analysis (IPA). Results: In the OCI-AML3 cell line, resazurin analysis demonstrated that combining CPI203 with nutlin-3 was potently synergistic in decreasing viable cells for a 1:12.5 (mean CI=0.07) and 1:25 ratio (mean CI=0.299), and synergistic for a 1:50(mean CI=0.44) and 1:100(mean CI= 0.66) ratios. There was no benefit in using the combination treatment on the p53 mutated cell lines. Apoptosis was enhanced at least 1.5 fold (median 1.7, range 1.5-2.65) by the drug combination versus the single agents, in the panel of p53 wild type cell lines tested. Analysis of whole genome RNA seq on OCI-AML3 treated cells, showed that genes up-regulated by the combination of CPI203 and nutlin-3, had a thirty-fold enrichment for p53 signalling (FDR (<0.05). Down-regulated genes were enriched for FOXM1-dependent cell cycle progression genes. To evaluate the combination in vivo, we used a Trib-2 driven primary AML where leukemogenesis is induced through inhibition of C/EBPα. Myeloblasts were transduced with GFP on the same retroviral construct asTrib-2 for disease tracking. Treatment was commenced in all mice (n=40), post confirmation of disease engraftment. Three mice from each treatment group were sacrificed after 48hrs and cells sorted for GFP to perform RNA seq in this in vivo setting. After 21 days of treatment all mice were sacrificed (n=27, one vehicle control succumbed to disease 15 days post engraftment). End of treatment results (primary read out was the GFP% which equates to the blast%) demonstrated superior in vivo efficacy of dual inhibition of MDM2 and BET in comparison with controls in eradicating AML, p<0.0001, (see figure). Importantly, normal haematopoiesis was spared - as evidenced by normal full blood counts and comparable myeloid, B-cell and T-cell populations with our C57bl6 wild type controls. RNA seq of the murine blasts revealed that many more genes significantly (FDR<0.05) changed expression in the combination treated mice than single agent treated mice. The p53 pathway was the most common up-stream regulator of genes changing expression post combination treatment, p<0.0001. The combination affected many more genes in the p53 pathway than RG7112 alone (120 genes versus 20 genes respectively), in line with our in vitro results. Conclusion: This combination of BET and MDM2 inhibition is effective and superior to single agent therapy on all p53 wild type AMLs tested, in vitro and in vivo. In both contexts this is associated with potentiating the p53 response and could be relevant to many patients with p53 wild type AML. Figure 1. Figure 1. Disclosures Latif: Novartis: Honoraria. Copland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Systems Biology Analysis Identifies Targetable Vulnerability Networks to Proteasome Inhibitors in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 950-950
Author(s):  
Mark B. Meads ◽  
Rafael Renatino Canevarolo ◽  
Praneeth Reddy Sudalagunta ◽  
Paula S. Oliveira ◽  
Dario M. Magaletti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Single Agent ◽  
Proteasome Inhibitors ◽  
In Vivo Models ◽  
Viability Assay

Abstract Proteasome inhibitors (PI) such as bortezomib and carfilzomib are critical components of anti-multiple myeloma (MM) therapy, yet all MM patients eventually develop refractory disease. We developed a non-biased method to identify and validate dysregulated pathways associated with PI-resistance in myeloma by combining RNAseq data from 522 MM patient specimens obtained from our Total Cancer Care/M2Gen/ORIEN network at Moffitt Cancer Center with paired ex vivo sensitivity to PIs and kinase inhibitors (KI). Dimensionality reduction analysis (t-SNE) and Fuzzy C-means was used to identify 422 clusters of genes that co-express in individual patients, and Gene Set Enrichment Analysis (GSEA) was used to identify clusters with gene expression patterns that correlated with PI sensitivity. Using publicly curated databases and in silico integrative analyses, we built protein-protein interaction networks to identify putative transcription factors, corresponding master regulators (kinases), and candidate KIs to promote PI sensitization. This systems biology approach identified a Chk1-Cdk1-Plk1 circuit associated with PI-resistance and also found 21 additional kinases (of 501 expressed in our cohort's kinome) that could be targeted to re-sensitize PI-resistant MM, which we confirmed in cell lines, specimens from relapsed patients, and two in vivo models. A panel of paired isogenic PI-resistant and sensitive MM cell lines were differentially screened to find kinases associated with PI-resistance using activity-based protein profiling (ABPP) and KI activity measured by high-throughput viability assay. The MM cell lines 8226 and U266, along with their drug resistant counterparts 8226-B25 and U266-PR, were grown in mono-culture for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe. Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer) to identify 85 kinases out of a total of 715 proteins in 8226-B25 MM cells and 35 kinases out of a total of 688 proteins in U266-PR MM cells that were preferentially enriched by 2-fold change compared to parental cell lines. Twenty-four kinases were commonly activated among PI-resistant cell line pairs and were screened in PI-resistant myeloma lines using a label-free, high throughput viability assay that simulates the tumor microenvironment. Three KIs targeting Plk1 (volasertib and GSK461364) and Cdk1/5 (dinaciclib) consistently maintained LD50s in the low-nanomolar range and induced caspase-3 activation in four PI-resistant MM cell lines: 8226-B25, U266-PR, ANBL-6-V10R, and Kas6-V10R. Twenty-four kinases each were identified by RNAseq/ex vivo PI sensitivity of MM specimens and ABPP of PI-resistant/sensitive MM cell line pairs. Of these, 7 kinases were identified by both methods: Cdk1, Chk1, Plk1, ILK, Syk, PKA, and p70S6K. Several KIs targeting Cdk1, Plk1, ILK, DNAPK, Syk, MKK7, Nek2, and mTOR identified in patient specimen or cell-line screens showed single agent activity in MM patient bone marrow specimens purified by a CD138 affinity column. Among these, inhibitors to Cdk1, ILK, mTOR, and Plk1 showed the most activity in patient specimens with an average 96h LD50 of 25 nM (n=56), 2.4 uM (n=42), 2.7 uM (n=57) and 3.8 uM (n=53), respectively. Six KIs targeting Plk1, ILK, Syk, MKK7, Nek2 and MARK3 were synergistic with carfilzomib in 20 patient specimens and maintained or improved ex vivo activity in relapsed refractory MM (RRMM) specimens. Volasertib, which targets Plk1, was the most synergistic with carfilzomib of all KIs tested in patient specimens and was further validated in two in vivo models: a NSG/U266 xenograft model of PI resistance and the syngeneic C57BL/6-KaLwRij/5TGM1 immunocompetent model. Volasertib significantly increased survival and reduced tumor burden in both models as a single agent, and was more effective versus PI-resistant tumors compared to PI-sensitive counterparts. Our pharmaco-proteomic screen, coupled with rich gene expression data from patients identified Plk1 as a target critical to MM survival in the context of acquired PI resistance and represents a unique workflow to find tumor vulnerabilities that arise during therapy. We anticipate that these data will also produce a critical path for the personalized allocation of therapy to maximize efficacy and minimize the use of ineffective therapies in RRMM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma

2021 ◽  
Author(s):  
Yu-bo Zhou ◽  
Yang-ming Zhang ◽  
Hong-hui Huang ◽  
Li-jing Shen ◽  
Xiao-feng Han ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Targets ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Preclinical Study ◽  
Parp Cleavage ◽  
Rpmi 8226 ◽  
Ongoing Phase

AbstractHDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%–35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

scholarly journals Experimental Models for Studying HPV-Positive and HPV-Negative Penile Cancer: New Tools for An Old Disease

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 460
Author(s):  
Beatriz Medeiros-Fonseca ◽  
Antonio Cubilla ◽  
Haissa Brito ◽  
Tânia Martins ◽  
Rui Medeiros ◽  
...  
Keyword(s):  
Mouse Models ◽  
Cell Lines ◽  
Penile Cancer ◽  
Hpv Infection ◽  
Experimental Models ◽  
In Vivo Models ◽  
Recent Developments ◽  
Key Aspects

Penile cancer is an uncommon malignancy that occurs most frequently in developing countries. Two pathways for penile carcinogenesis are currently recognized: one driven by human papillomavirus (HPV) infection and another HPV-independent route, associated with chronic inflammation. Progress on the clinical management of this disease has been slow, partly due to the lack of preclinical models for translational research. However, exciting recent developments are changing this landscape, with new in vitro and in vivo models becoming available. These include mouse models for HPV+ and HPV− penile cancer and multiple cell lines representing HPV− lesions. The present review addresses these new advances, summarizing available models, comparing their characteristics and potential uses and discussing areas that require further improvement. Recent breakthroughs achieved using these models are also discussed, particularly those developments pertaining to HPV-driven cancer. Two key aspects that still require improvement are the establishment of cell lines that can represent HPV+ penile carcinomas and the development of mouse models to study metastatic disease. Overall, the growing array of in vitro and in vivo models for penile cancer provides new and useful tools for researchers in the field and is expected to accelerate pre-clinical research on this disease.

scholarly journals BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽  
2018 ◽  
Vol 3 (6) ◽  
pp. e000387 ◽  
Author(s):  
Chiara Tarantelli ◽  
Elena Bernasconi ◽  
Eugenio Gaudio ◽  
Luciano Cascione ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Antitumour Activity ◽  
Single Agent ◽  
Treatment Strategies ◽  
Bet Inhibitor ◽  
Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

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