scholarly journals CC-92480 Enhances Cell-Autonomous Cytotoxicity through Blockade of G 2/M Transition When Combined with Bortezomib/Dexamethasone in Pre-Clinical Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2669-2669
Author(s):  
Chad C Bjorklund ◽  
Michael Amatangelo ◽  
Jian Kang ◽  
Hsiling Chiu ◽  
Archana Mukhopadhyay ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Single Agent ◽  
Gene Set Enrichment Analysis ◽  
Brdu Incorporation ◽  
Publicly Traded ◽  
Inhibition Of Proliferation ◽  
Clinical Pharmacokinetics ◽  
Apoptotic Induction ◽  
Privately Held

Abstract Background: Pomalidomide (POM) is an established agent in relapsed/refractory (R/R) multiple myeloma (MM). CC-92480, a novel cereblon E3 ligase modulator (CELMoD ®) agent, is being investigated in R/R MM patients in combination with the proteasome inhibitor (PI) bortezomib (BTZ) and steroid dexamethasone (DEX) (NCT03374085/NCT03989414). Previously, we showed mechanistic synergy of POM/BTZ/DEX in MM cell line models (Bjorklund et al). Here we analyzed the cell autonomous cytotoxic activities of CC-92480 or POM alone and in combination with BTZ/DEX to compare and differentiate their mechanisms of action (MOA). Results: Comparative analysis of the anti-proliferative activity against H929 and MM1.S cell lines revealed that CC-92480 demonstrated a more potent inhibition of proliferation by 100-fold lower dose compared to POM. Combination experiments utilizing a titration of POM or CC-92480 in combination with a 1 hr BTZ pulse, to mimic the clinical pharmacokinetics (+/- DEX co-treatment) showed an enhancement of antiproliferative capacity in both doublet and triplet combinations compared to single agents. Combination indices for POM/BTZ/DEX or CC-92480/BTZ/DEX resulted in values <1 for most combinations indicating a synergistic effect. Additionally, POM or CC-92480 in combination with BTZ or DEX, or in triplet combinations increased induction of apoptosis (>90% for each triplet compared to POM (20%) and CC-92480 (40%). Flow cytometric analysis of Aiolos and Ikaros protein level in MM cells treated with POM/BTZ/DEX or CC-92480/BTZ/DEX resulted in a slight kinetic delay in substrate depletion at early time points (1-4 hr), where the effect is less apparent with CC-92480, and indistinguishable at 24 hr compared to single agent POM or CC-92480 in the clinically relevant concentrations. We performed transcriptomic analyses of H929 cells treated with POM/BTZ/DEX or CC-92480/BTZ/DEX for 24 hrs to identify key pathways responsible for the observed synergistic combination effect. Common pathways dysregulated by POM or CC-92480 included previously identified interferon, protein homeostasis and proliferation gene sets. Gene set enrichment analysis (GSEA) showed many significant pathway differences when comparing the triplets, including general cell cycle progression, cell division and chromatin segregation. Interestingly, genes involved in negative regulation of G 2/M transition were identified as one of the most significant differences between POM/BTZ/DEX and CC-92480/BTZ/DEX. To understand how these pathways contributed to cell cycle effects and apoptosis, we assessed DNA fragmentation by TUNEL in conjunction with cell cycle flow cytometry to examine cell cycle specific apoptotic induction. Temporal assessment (6, 12, 18, 24, and 48 hr treatments) demonstrated accumulation of BrdU incorporation in all cell cycle phases when treated with POM/BTZ/DEX or CC-92480/BTZ/DEX indicating cell death was occurring within all phases. However, there was a marked enhancement of G 2/M BrdU incorporation (80% vs. 40% of G 2/M population) at 18-24 hr when treated with CC-92480/BTZ/DEX compared to POM/BTZ/DEX, or other single agent treatments. Additionally, G 2/M transition-dependent cyclins A and B were shown to be dysregulated by CC-92480. These data indicate that CC-92480 potentiates a G 2/M arrest in combination with BTZ in MM cells. Conclusions: These results demonstrate that CC-92480 alone or in combination with BTZ/DEX elicits a more potent cytotoxic effect on MM cells compared to POM. Importantly, the combination of either POM or CC-92480 with a PI, like BTZ, does not appreciatively affect single agent MOA. We have also identified a key differentiating mechanism of cell autonomous activity for CC-92480 in combination with BTZ/DEX where MM cells enhance apoptotic induction at the G 2/M stage compared to POM. Clinically, this added mechanistic difference suggests a more cytotoxic response in patients treated with CC-92480/BTZ/DEX compared to POM/BTZ/DEX. Disclosures Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kang: BMS: Current equity holder in publicly-traded company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Hagner: BMS: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

scholarly journals H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2200-2200
Author(s):  
Pan Zhou ◽  
Mengyu Xiao ◽  
Yanliang Bai ◽  
Huixia Cao ◽  
Weiping Yuan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Mapk Signaling ◽  
Gene Set Enrichment Analysis ◽  
Luciferase Reporter ◽  
Brdu Incorporation ◽  
Down Regulation

Abstract In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma. To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM. We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM. To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c-Myc using 10058-F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM. Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. Disclosures No relevant conflicts of interest to declare.

Dehydroandrographolide Inhibits Osteosarcoma Cell Growth and Metastasis by Targeting SATB2-mediated EMT

2019 ◽  
Vol 19 (14) ◽  
pp. 1728-1736
Author(s):  
Xuefeng Liu ◽  
Yonggang Fan ◽  
Jing Xie ◽  
Li Zhang ◽  
Lihua Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Wound Healing ◽  
Cell Growth ◽  
Brdu Incorporation ◽  
Migration And Invasion ◽  
Therapeutic Drug ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Inhibition Of Proliferation ◽  
Predominant Component

Background:The 12-hydroxy-14-dehydroandrographolide (DP) is a predominant component of the traditional herbal medicine Andrographis paniculata (Burm. f.) Nees (Acanthaceae). Recent studies have shown that DP exhibits potent anti-cancer effects against oral and colon cancer cells.Objective:This investigation examined the potential effects of DP against osteosarcoma cell.Methods:A cell analyzer was used to measure cell viability. The cell growth and proliferation were performed by Flow cytometry and BrdU incorporation assay. The cell migration and invasion were determined by wound healing and transwell assay. The expression of EMT related proteins was examined by Western blot analysis.Results:In this study, we found that DP treatment repressed osteosarcoma (OS) cell growth in a dose-dependent manner. DP treatment significantly inhibited OS cell proliferation by arresting the cell cycle at G2/M phase. In addition, DP treatment effectively inhibited the migration and invasion abilities of OS cells through wound healing and Transwell tests. Mechanistic studies revealed that DP treatment effectively rescued the epithelialmesenchymal transition (EMT), while forced expression of SATB2 in OS cells markedly reversed the pharmacological effect of DP on EMT.Conclusion:Our data demonstrated that DP repressed OS cell growth through inhibition of proliferation and cell cycle arrest; DP also inhibited metastatic capability of OS cells through a reversal of EMT by targeting SATB2. These findings demonstrate DP’s potential as a therapeutic drug for OS treatment.

Activity of Tyrosine Kinase Inhibitors in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4804-4804
Author(s):  
Reinhold Munker ◽  
Cory Cordova ◽  
Paula Polk ◽  
Charles V. Wendling ◽  
Amanda W. Sun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Single Agent ◽  
Calf Serum ◽  
Gene Expression Pattern ◽  
Inhibition Of Proliferation ◽  
Short Term Exposure

Abstract Tyrosine kinase inhibitors (TKIs) have been successfully introduced for the treatment of cancer. Imatinib, dasatinib and nilotinib target bcr/abl and were found to induce molecular remissions in chronic myeloid leukemia. Imatinib has also been found to be active in other malignancies like gastrointestinal stromal tumors. Sunitinib and sorafenib are multi-targeted tyrosine kinase inhibitors and so far, have shown activity against renal cell carcinoma and other cancers. Gefitinib targets the tyrosine kinase of epidermal growth factor receptor and has been found to be active against some cases of non-small cell lung cancer. There is circumstantial evidence that tyrosine kinases and their receptors (e.g. VEGF, IGF-1 and FGFR3) are active in multiple myeloma. In order to develop new treatments for multiple myeloma (MM), we tested several currently available TKIs for their activity against MM cell lines. Materials and methods: The a cell lines MC/CAR, ARH77, RPMI 8226, ARP1, JJN3, MM1S, and INA-6 were treated with various concentrations of TKIs and analyzed for cell growth in liquid culture, proliferation, apoptosis, and gene expression pattern screening 14,500 genes using U133A_2 arrays. Results: Imatinib, nilotinib, dasatinib, gefitinib induced cytotoxicity in most cases at high concentrations (50% inhibitory concentration ≥ 100 μMol), whereas sunitinib and sorafenib were active at lower concentrations (50% IC 1– 5 μMol). The cytoxicity was observed early (within 4 to 24 hours of exposure) and involves apoptosis. Interleukin-6 did not offer protection against the cytotoxicity of sorafenib or sunitinib, however the inhibition of proliferation was more pronounced in low fetal calf serum (2.5 versus 10%). A short-term exposure of the myeloma cell line MM1S to 10 μMol sorafenib resulted in more than 2 fold changes in 283 genes or sequences (175 up, 108 down). If only 10 fold changes are considered, 21 genes or sequences were upregulated (mainly enzymes, regulators and ligands) and 11 downregulated (mainly regulatory proteins, among them IL6 signal transducer). Conclusion: We found that the multitargeted TKIs sorafenib and sunitinib are active in vitro against multiple myeloma. We plan to investigate patient samples, and to elucidate the targets and the mechanisms of action. Our data will support clinical trials both as single agent and in combination with other drugs like bortezomib, thalidomide, alkylators and ionizing radiation.

Adenosine A2A and Beta-2 Adrenergic Receptor Agonism: Novel Selective and Synergistic Multiple Myeloma Targets Discovered through Systematic Combination Screening

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 384-384
Author(s):  
Richard J. Rickles ◽  
Laura Pierce ◽  
Thomas Giordano ◽  
Winnie F. Tam ◽  
William Avery ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
High Throughput ◽  
Adrenergic Receptor ◽  
Molecular Mechanisms ◽  
Single Agent ◽  
Inhibition Of Proliferation ◽  
Cgs 21680 ◽  
Beta 2 ◽  
Adenosine A2a

Abstract Using a high throughput combination screening strategy, we have discovered that agonism of either adenosine A2A receptors (A2A) or beta-2 adrenergic receptors (bAR) demonstrate significant, synergistic, anti-proliferative effects in preclinical Multiple Myeloma (MM) models. Using quantitative synergy analysis, we observe that A2A and bAR agonists have significant anti-proliferative effects in a broad panel of 10 MM cell lines when combined with each other or with standard MM agents. Individual A2A agonists CGS-21680 and HE-NECA inhibited proliferation 25–80% with EC50s ranging from 2–20 nM. Individual bAR agonists salmeterol and formoterol inhibited proliferation 35–75% with EC50s ranging from 10–30 pM. Potent, highly synergistic, inhibition of proliferation, up to 95%, was demonstrated with combinations of A2A or bAR agonists and multiple agents including dexamethasone, lenalidomide, bortezomib, melphalan, doxorubicin, HDAC inhibitors and HSP90 inhibitors at clinically relevant concentrations. These combinations exceeded Loewe additivity, and demonstrated both substantial increases in efficacy over maximal single agent levels as well as significant potency shifting with many combination indices (CIs) in the range of 0.1 to 0.3. Synergistic anti-proliferative effects were observed broadly across several MM cell lines and when using cell lines unresponsive to standard MM drugs, e.g. A2A agonists CGS-21680 and HE-NECA in combination with dexamethasone inhibited 75–85% of the proliferation of EJM, and MOLP-8 dexamethasone-insensitive cell lines as compared to 35–60% for the single agent A2A agonists. The selective A2A antagonist SCH58261 but not A1, A2B and A3 selective antagonists DPCPX, MRS1754 and MRS1523 blocked the synergy and antiproliferative activity of HE-NECA, demonstrating that the effect is mediated via the A2A receptor. siRNA directed against adenosine and adrenergic receptor isoforms, caused a concomitant reduction in the antiproliferative effects of HE-NECA and salmeterol. Synergy (CI<0.4) observed between A2A and bAR agonists suggested that while both these targets signal through Gs coupled signaling pathways, the two targets contribute to the antiproliferative effect via distinct molecular mechanisms. Anti-proliferative effects occurred through a synergistic induction of apoptosis. Combinations of either agonist with dexamethasone demonstrate 50–75% Annexin V positive MM.1S cells after 24 hours treatment whereas single agents show less than 10%. The activity, synergy and selectivity of A2A and bAR combinations were observed in xenograft models of MM. SCID CB17 mice received subcutaneous inoculation of RPMI-8226 cells and once tumors were palpable, mice were treated with vehicle, bortezomib (0.5 mg/kg IV Q3D), salmeterol (10 mg/kg s.c QD) or the combination of both agents. After 19 days of treatment, the combination showed significantly greater reduction in tumor volume than either of the single agents alone (70% vs. 34%; p<0.05, ANOVA). High throughput combination screening facilitated the discovery of two novel and related classes of drug targets with highly synergistic and selective anti-tumor activity in MM. These preclinical data provide a strong rationale for the investigation of A2A and bAR agonists in the treatment of MM.

scholarly journals Pathway Analysis Provides Mechanistic Insights into Navtemadlin (KRT-232) Activity and Synergy with Decitabine in Acute Myeloid Leukemia Preclinical Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2232-2232
Author(s):  
Jin Wang ◽  
Venu Valmeekam ◽  
Kaijin Wu ◽  
Jiang F. Zhong ◽  
Andres Stucky ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
Combination Treatment ◽  
Single Agent ◽  
Rna Seq ◽  
Publicly Traded ◽  
Apoptotic Pathway ◽  
Ecm Remodeling ◽  
P53 Signaling ◽  
Privately Held

Abstract Background: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, restores p53 activity to drive apoptosis of cancer cells in TP53 WT malignancies through the intrinsic (mitochondrial) pathway. Navtemadlin is being evaluated in a phase 3 trial in relapsed or refractory myelofibrosis, and in phase 1b/2 trials in various hematologic malignancies and solid tumors. Because p53 is infrequently mutated and MDM2 is often overexpressed in acute myeloid leukemia (AML), navtemadlin represents a rational therapeutic candidate (Prokocimer et al. Blood. 2017). Synergy between navtemadlin and the hypomethylating agent decitabine has been demonstrated in preclinical AML models (Canon et al. AACR. 2016), although the mechanisms mediating this effect remain unclear. The present study aims to identify mechanisms of navtemadlin-mediated apoptosis within AML cell lines, timing of pathway activation, and effects of combination treatment with navtemadlin + decitabine. Methods: MOLM13 or MV-4-11 AML cell lines were treated with a vehicle control, 0.5-1.0 µM navtemadlin, 1.0 µM decitabine, or with a combination of navtemadlin + decitabine. Cells were pretreated with decitabine for 42-48 hours followed by navtemadlin + fresh decitabine for 0, 5, 10, or 24 hours, and were then harvested for RNA-Seq and immunoblotting analyses of apoptotic pathway proteins. Gene set enrichment analysis (GSEA) and signaling pathway impact analysis (SPIA) were performed on RNA-Seq data to determine the most differentially regulated pathways (using KEGG curated signaling pathways) between controls, single-agent navtemadlin or decitabine, or combination treatment. Protein expression analyses were conducted to validate RNA-Seq data. Results: GSEA of MOLM13 and MV-4-11 cells at 10 hours showed that, relative to controls, several signaling pathways were differentially regulated to a statistically significant level (P≤0.05) with navtemadlin alone, including activation of apoptosis and p53 signaling pathways, and suppression of DNA replication and cell cycle pathways, in line with previous reports (Figure 1a). In MOLM13 cells treated with single-agent decitabine, lipid and proteoglycan-related signaling pathways were activated, whereas several metabolic pathways were suppressed. With combination treatment in both cell lines, unique activated pathways relative to controls included immune-related and cancer-signaling pathways; and unique suppressed pathways included base excision repair and cellular senescence pathways. SPIA analysis of both cell lines at 10 hours produced higher perturbation scores for apoptosis and p53 signaling pathways with combination treatment vs navtemadlin alone (Figure 1b), indicating more robust pathway activation. Additionally, tumor microenvironment (TME) and extracellular matrix (ECM) remodeling pathways showed more robust perturbation when combined with navtemadlin vs decitabine alone. At 24 hours, only SPIA was conducted, which showed less apoptotic pathway induction relative to 10 hours in both cell lines, suggesting time-dependent signaling effects. Cellular senescence pathways were suppressed, whereas central carbon metabolism and HIF-1 signaling were induced by single-agent navtemadlin relative to controls in both cell lines, with combination treatment further enhancing these effects. Combination treatment also induced significant changes in the expression of ECM remodeling genes including MMP9, MMP2, and ITGB7. Protein expression analyses in both cell lines confirmed early upregulation of key apoptosis regulators including p53, p21, PUMA, cleaved caspase-3 and cleaved-PARP with navtemadlin alone, and the DNA-damage response protein, yH2AX, with decitabine alone. The combination enhanced the induction of p53, p21, and yH2AX vs controls or single agents alone. Conclusion: Initial results from this study suggest that navtemadlin + decitabine enhanced apoptosis and p53 signaling pathways to a greater degree than either single agent in AML cell lines. Combination treatment also induced several unique DNA damage response, ECM-remodeling, and TME-related pathways. Activation of both proapoptotic and tumor-stromal interaction pathways suggest a unique mechanism of navtemadlin + decitabine for inhibiting AML cell growth. Figure 1 Figure 1. Disclosures Valmeekam: Telios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company, Other: travel, accommodations, expenses. Canon: Oncovalent Therapeutics: Current Employment; Amgen, Inc.: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Krejsa: Acerta Pharma: Current holder of individual stocks in a privately-held company; AstraZeneca: Current equity holder in publicly-traded company; Seattle Genetics: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Kartos Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Other: travel, accommodations, expenses. Kelly: AstraZeneca: Consultancy; Bayer: Speakers Bureau; Verastem: Consultancy; Amgen: Consultancy; Berkley Lights: Current equity holder in publicly-traded company; Denovo Biopharma: Consultancy; Takeda: Consultancy; Sanofi-Aventis: Consultancy; Novartis: Speakers Bureau; Janssen: Speakers Bureau; Agios: Current equity holder in publicly-traded company; Celgene: Speakers Bureau; Epizyme: Speakers Bureau; Pharmacyclics: Speakers Bureau; Karyopharm: Speakers Bureau; Gilead: Speakers Bureau. OffLabel Disclosure: Yes, navtemadlin (KRT-232) is an investigational small molecule inhibitor.

scholarly journals Preclinical Activity of Selective SYK Inhibitors, Entospletinib and Lanraplenib, Alone or Combined with Targeted Agents in Ex Vivo AML Models with Diverse Mutational Backgrounds

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3356-3356
Author(s):  
Melinda A. L. Day ◽  
Philipp Sergeev ◽  
Caroline A. Heckman ◽  
Anna Schinzel ◽  
Nikolaus D. Obholzer ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Viability ◽  
Research Funding ◽  
Ex Vivo ◽  
Single Agent ◽  
Clinical Activity ◽  
Publicly Traded ◽  
Aberrant Expression ◽  
Syk Inhibitor ◽  
Privately Held

Abstract Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that mediates integrin and Fc receptor signaling in myeloid cells. SYK has been implicated as an oncogenic driver in acute myeloid leukemia (AML) with aberrant expression of HOXA9 and MEIS1 and cooperates with FLT3 internal tandem duplication to drive leukemogenesis. The oral SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity in HOXA9/MEIS1 driven AML and is currently being investigated in a phase 3 trial of previously untreated patients with nucleophosmin1-mutated (NPM1 mut) AML. Lanraplenib (LANRA) is a next generation oral SYK inhibitor with potency and selectivity comparable to ENTO. In healthy volunteers and patients with autoimmune disease, LANRA has shown pharmacokinetic properties that compare favorably with ENTO. To support the clinical development of LANRA for the treatment of AML, ex vivo treatment of patient-derived AML cells was used to compare its activity to that of ENTO, both as a single-agent and in combination with other AML therapies. First, ENTO and LANRA single-agent activities were evaluated in peripheral blood-derived blasts from 15 AML patients, representing different mutational backgrounds including NPM1, FLT3, PTPN11, and NRAS mutations. AML cells were seeded into 96 well plates and treated with ENTO and LANRA for 6 days. Comparable effects on viability were observed across the 15 models with the 2 compounds, and in 11 of the models, the half maximal inhibitory concentration (IC 50) values were within 2-fold of each other. ENTO had a slightly lower IC 50 value than LANRA in the FLT3-mutated models possibly due to the direct FLT3 inhibitory activity of ENTO. Next, we tested the activity of ENTO and LANRA ex vivo in bone marrow-derived AML blasts from 29 AML patients representing diverse mutational backgrounds, including NPM1, IDH1, FLT3, and RAS mutations as well as MLL rearrangements. The models were treated for 9 days with either ENTO or LANRA, and viability was assessed using Annexin V and 7-aminoactinomycin D staining. Again, ENTO and LANRA showed comparable effects on cell viability with no significant differences between the compounds when compared across the different mutational backgrounds. Both studies suggest the potential for anti-leukemic activity in several different genetically defined subsets of AML. Matrix combination assays were performed by combining ENTO or LANRA with either cytarabine (NPM1 mut), gilteritinib (FLT3 mut), or trametinib (RAS mut) with cell viability and death assessed after a 3-day incubation period. Increased cell death in an additive manner was observed in all combinations tested, with results for ENTO and LANRA being similar, indicating the utility of both compounds in combinatorial treatment paradigms. These results support the clinical evaluation of LANRA in genetically defined subsets of AML. A phase 1b/2 study of LANRA in combination with the selective FLT3 inhibitor gilteritinib, in patients with relapsed or refractory FLT3 mut AML is planned for the end of this year. Disclosures Day: Cyteir Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding. Schinzel: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Obholzer: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lin: Kronos Bio, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Saffran: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Pre-Clinical and Clinical Immunomodulatory Effects of Pomalidomide or CC-92480 in Combination with Bortezomib in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1613-1613
Author(s):  
Chad C Bjorklund ◽  
Michael Amatangelo ◽  
Hsiling Chiu ◽  
Jian Kang ◽  
Tiziana Civardi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Granzyme B ◽  
Target Cells ◽  
Publicly Traded ◽  
Dose Dependent ◽  
Privately Held

Abstract Background: Pomalidomide (POM) is an established agent in relapsed/refractory (R/R) multiple myeloma (MM) with direct cytotoxicity against MM cells and immunostimulatory activities in multiple cell types including T cells and NK cells. CC-92480 is a novel Aiolos/Ikaros degrading cereblon E3 ligase modulator (CELMoD ®) agent is currently being investigated in combination with the proteasome inhibitor (PI) bortezomib (BTZ) and corticosteroid dexamethasone (DEX), or with DEX only in R/R MM (CC-92480-MM-002 and CC-92480-MM-001). Previous results indicate that triplet combination of POM/BTZ/DEX may enhance some T, B and NK cell subpopulations, overcoming immunosuppression when compared to BTZ/DEX-only treated patients (Rao et al, 2019). Mechanisms of action (MOA) of CC-92480- and POM-mediated substrate depletion occurs via ubiquitination and proteasome degradation, where BTZ has been speculated as potentially antagonistic as a PI. Here, we report pre-clinical and clinical observations of an immune MOA of CC-92480 or POM in combination with BTZ. Results: To mimic the clinical pharmacokinetics, BTZ was utilized as a high-dose pulse method alone and in combination with POM or CC-92480, followed by flow cytometric measurements of Aiolos and Ikaros protein abundance in healthy donor (HD) T cells. The addition of BTZ modestly delayed CRBN-dependent substrate depletion compared to single agent POM or CC-92480; however, this effect was only apparent at early time points (1-6 hr) where the effect was negligible by 24 hr. To understand the functional implications of BTZ combination, we conducted CD3-stimulated PBMC-mediated cytotoxicity assay against H929 MM target cells in a co-culture model. The efficiency of POM or CC-92480 induced PBMC-mediated killing in a dose dependent manner (~65% increase compared to DMSO) were similar at a 100-fold lower dose range of CC-92480 compared to POM, with the effect not being altered by co-treatment with BTZ. These data were replicated with a POM or CC-92480 treated supernatant stimulation of purified NK cells co-culture, which induced an 80% reduction in target cell viability with the BTZ combination having no negative effects on CELMoD-mediated activity. Cytokine analysis on PBMC supernatants treated with either POM or CC-92480 in the absence or presence of BTZ-pulse showed a dose-dependent increase in IL-2 (>2.4-fold) and Granzyme B (>3.1-fold), which were not impacted by BTZ co-treatment. As a secondary readout on activation status, we measured multiple signaling molecules and activation markers on the cell surface of T and NK cell subsets in CD3 stimulated HD PBMCs treated with dose-dependent POM or CC-92480 with or without co-treatment of BTZ. Compared to DMSO controls, elevated expression levels of CD25 (IL2RA), CD278 (ICOS), Granzyme B, CD134 (OX40R) and HLA-DR were observed with both POM and CC-92480 on CD4, CD8 and NK cells demonstrating a CELMoD-mediated increase in immune activation. These effects were not impacted by the co-treatment of BTZ. Examination of peripheral blood samples from MM patients enrolled in the CC-92480-MM-001/002 (NCT03374085/NCT03989414) clinical trials revealed that CC-92480 promoted potent immunomodulation when administered in combination with DEX and with BTZ/DEX. These data included increased numbers of activated and central memory T cells, as well as increased Ki67+ proliferating T and NK cell populations compared to samples collected during the screening period before any drugs had been administered, consistent with earlier observation of POM in combination with BTZ/DEX treated patients. Conclusions: Taken together, these data demonstrate that POM and CC-92480 are potent immunomodulatory agents with enhanced induction of PBMC and NK mediated cell killing of MM tumor cells and activation of T and NK cells, at 100-fold lower concentrations of CC-92480 compared to POM. Additionally, we showed that combination with BTZ in preclinical assays and in the clinical setting did not antagonistically affect the immunostimulatory ability of POM or CC-92480. Disclosures Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kang: BMS: Current equity holder in publicly-traded company. Civardi: Bristol Myers Squibb: Current Employment. Katz: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Maciag: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hagner: BMS: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Bahlis: Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Richardson: Oncopeptides: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

scholarly journals Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2658-2658
Author(s):  
Aarif Ahsan ◽  
Ann Polonskaia ◽  
Chih-Chao Hsu ◽  
Chad C Bjorklund ◽  
Maria Ortiz Estevez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
High Risk ◽  
Cell Lines ◽  
Publicly Traded ◽  
Chip Sequencing ◽  
Chromosome 1Q ◽  
Cycle Arrest ◽  
Super Enhancer ◽  
Sequencing Studies

Abstract Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability. Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines. Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P<0.001) and enhanced apoptosis in MM cell lines. BRD4-ChIP sequencing studies revealed that the expression of CKS1B was regulated by super-enhancer (SE) associated elements. As expected, two BRD4 inhibitors, JQ1 and CC-90010 and inducible BRD4 shRNAs downregulated the expression of CKS1B resulting in decreased proliferation, cell cycle arrest and apoptosis in MM cell lines. Furthermore, MM cell lines harboring chromosome 1q gain/amp showed higher sensitivity to BRD4 inhibition compared to cell lines with normal 1q copy number. Mechanistic studies revealed that BRD4inh and BRD4 shRNAs downregulated the expression of Aiolos and Ikaros in MM cell lines. Interestingly, Aiolos ChIP-sequencing studies demonstrated the binding of Aiolos at the transcriptional start sites of CKS1B with the transcriptional activation mark. The immunomodulatory agent (IMiD ®) pomalidomide (Pom) transcriptionally downregulated CKS1B in Pom-sensitive cells downstream of Aiolos, Ikaros degradation. Based on these mechanisms, IMiD agents, lenalidomide, Pom and the novel Cereblon E3 ligase modulating degrader (CELMoD ®) agent CC-92480 in combination with BRD4inh promoted a synergistic decrease in proliferation, cell cycle arrest and increase in apoptosis in both Pom-sensitive and -resistant cell lines. The combination of IMiD or novel CELMoD agent with BRD4inh also promoted deeper downregulation of CKS1B, Aiolos, Ikaros, c-Myc and survivin proteins with enhanced levels of apoptotic marker cleaved Caspase 3 as compared to single agents alone. Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients. Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. Figure 1 Figure 1. Disclosures Ahsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

The Co-Occurrence of MAF Translocations in RAS Mutated Multiple Myeloma Confers Resistance to MEK Inhibition

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1138-1138 ◽  
Author(s):  
Ya-Wei Qiang ◽  
Shiqiao Ye ◽  
Yu Chen ◽  
Amy Buros ◽  
Frits van Rhee ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Mapk Pathway ◽  
Expression System ◽  
Single Agent ◽  
Ectopic Expression ◽  
Brdu Incorporation ◽  
Ras Mutation ◽  
Ic50 Value

Abstract Introduction Mutant NRAS and KRAS lead to the activation of the RAS/RAF/MEK/ERK pathway in approximately 50% of multiple myeloma (MM). Blocking this pathway with MEK1/2 inhibitors (MEKi) such as trametinib (Tram) is a therapeutic option but the response rate in MM varies between 30-50% (Heuck et al, Leukemia 2015). In MM it is unknown whether RAS mutation status correlates with sensitivity to Tram. The purpose of this study was to characterize factors which predict response to Tram and to identify mechanism mediating resistance. Methods We established the IC50 of Tram using MTT assays in 32 MM cell lines (HMCL) including 16 RAS mutant positive (RASm+), and 15 wildtype RAS (RASm-), and 1 BRAF mutant (BRAFm+) line which acted as a positive control. HMCLs were classified according to the IC50 value as sensitive (<0.05μM); intermediate sensitive (IMS) (0.05 and 10μM); and resistant (>10μM). All lines underwent immunoblotting for pERK at baseline and following treatment with serial concentrations of Tram to identify correlation of activation with sensitivity. BrdU incorporation analyzed by FACS was performed to determine the molecular action of Tram. A lentiviral mediated expression system was used to engineer a MAF overexpressing cell line in a RASm+ HMCL lacking MAF (MMRASm+MAF) and silencing MAF in two lines with co-occurring MAF and RASm+ (MMRASm+shMAF). The clinical characteristics of 84 relapsed RASm+ patients who received Tram either as a single agent or in combination with other anti-MM therapies were also examined. Results 6/16 (37.5%) of RASm+ HMCLs were sensitive to Tram, 5/16 (31.1%) were IMS and 5/16 (31.1%) resistant. There was no difference in sensitivity to Tram between KRASm+ (IC50 = 9.5μM, n = 11) and NRASm+ (IC50 =12.5μM, n=4, p=0.65). In contrast, 13/15 (87%) RASm- HMCLs were resistant to Tram. Mechanistically, Tram blocked cell cycle progression in Tram-sensitive RASm+ cells with an increase in G0/G1 phase (22.25%) and a decrease in S phase (16.76%) compared with untreated controls (p<0.01). These in-vitro findings correlated with our clinical experience where there was no difference in progression free or overall survival (OS) between KRASm+ (n=46) or NRASm+ (n=38) patients treated with Tram. Given that RASm+ lines exhibit a range of sensitivity (IC50 0.005-30μM), we further defined additional molecular modifiers that influence sensitivity by comparing KRASm+ alone with KRASm+ co-occurring with t(14;16) (n=4) or t(4;14) (n=3). The mean IC50 value of Tram (0.025μM) in 5 RASm+ was significantly lower than those with a co-occurring t(14;16) (IC50 =16μM; P<0.001) or a t(4;14) (IC50 =11.6μM, P<0.05). Among 15 RASm- HMCLs, the IC50 (37.5μM, n=9) of Tram was much higher in t(14;16) HMCL than in t(4;14) (IC50=9.16μM, n=3, P<0.01). A similar pattern was seen in patients, with a shorter OS when RASm+ co-occurred with t(14;16) or t(4;14) (n=17 OS=0.99yrs) than those with RASm+ alone (n= 67, OS=1.85yrs, P=0.01). These findings provide evidence that co-occurring MAF expression renders RAS+ MM less sensitive to MAKP inhibition. To determine if activation of MAPK pathway caused by RAS mutation was associated with sensitivity to MEKi, we assess baseline and post Tram treatment levels of p-ERK1/2, a downstream substrate of MEK1/2 activation. MM HMCLs displayed variable basal levels of pERK. There was no correlation between basal levels of pERK and RASm+, with the highest baseline levels of pERK being seen in HMCLs with a t(14;16) or t(14;20) and wildtype RAS, intermediate levels in RASm+ with a t(14;16) and lowest in RASm+ alone. The concentrations of Tram for complete inhibition of pERK were higher in HMCLs with co-occurring t(14;16) ( >or = 500 nM) than for RASm+ alone. To further confirm the ability of MAF to confer resistance of RASm+ cells to MEKi, we examined the sensitivity of MMRASm+/MAF to Tram.MTT assay showed that the IC50 of MM RASm+/MAF cells was 50% higher than that of MMEV cells (P <0.001). Similar results were observed in two other HMCLs with transient ectopic expression of MAF indicating that increase in MAF protein in RAS mutant cells reduces sensitivity to MEKi. In contrast, silencing of MAF in two RASm+ HMCLs significantly increased sensitivity (75% decrease in IC50) to Tram, compared with MMRASm+shCont (P<0.001). Conclusion Our results demonstrate that RASm+ MM is sensitive to Tram but the co-occurrence of t(14;16) leading to MAF overexpression confers resistance both in-vitro and in patients. Disclosures Morgan: Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

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.

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