Enhanced Multiple Myeloma Cell Research Articles

LncRNA MALAT1 silencing represses CXCL12-induced proliferation, invasion, and homing behavior in multiple myeloma by inhibiting CXCR4

ABSTRACT Background: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been identified in multiple myeloma (MM); its influence on the homing behavior in MM cells is unclear. This study elucidates the impact and mechanism by which MALAT1 affects the homing behavior in MM cells. Methods: Bone marrow was obtained from patients with MM. MALAT1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cluster of differentiation 138-purified (CD138) plasma cells were detected by qRT-PCR and Western blotting. MALAT1, CXCR4, and C-X-C motif chemokine ligand 12 (CXCL12) influences on MM cell viability, proliferation, and invasion was monitored by CCK-8, 5-Ethynyl-2'-deoxyuridine, and Transwell assays. qRT-PCR, Western blotting, and ELISA were utilized to detect homing effect-related proteins in MM cells, including intercellular adhesion molecule 1 (ICAM-1) and very late antigen-4 (VLA-4). Results: MALAT1 and CXCR4 were overexpressed in CD138-purified plasma cells from bone marrow of MM patients, associating with bone damage. MALAT1 upregulated CXCR4 in MM cells. MALAT1 overexpression enhanced MM cell viability, proliferation, and invasion, whereas CXCR4 silencing reversed them. CXCR4 silencing attenuated MALAT1 induction on ICAM-1 and VLA-4 expression in MM cells. CXCL12 upregulation intensified MM cell proliferation and invasion and ICAM-1 and VLA-4 expression in MM cells. MALAT1 silencing counteracted the impact of CXCL12. Conclusion: MALAT1 silencing may repress CXCL12 induction on MM cell proliferation, invasion, and homing behavior by inhibiting CXCR4. MALAT1 may be a promising target for MM treatment.

Dual therapeutic targeting of MYC and JUNB transcriptional programs for enhanced anti-myeloma activity

Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes.

E3 Ubiquitin Ligase TRIM21 Enhances Macrophage-Mediated Bortezomib Resistance By Inducing M2 Polarization in Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell hematologic tumor with an increasing incidence and has become the second most common hematologic malignancy after non-Hodgkin lymphoma. Macrophage is an abundant component of the MM bone marrow microenvironment and contributes to the enhancement of MM drug resistance. Our previous study found that the expression of the E3 ubiquitin ligase TRIM21 was reduced in MM cells, and MM cells with low expression of TRIM21 showed reduced sensitivity to Bortezomib (Bort). This study aims to investigate the role and mechanism of TRIM21 in macrophage-mediated MM Bort resistance. Objective ：Explore the effect and mechanism of TRIM21 in mediating multiple myeloma Bortezomib resistance. Methods ：1. Immunofluorescence to analyze the expression of TRIM21 on macrophages in bone marrow biopsy specimens from patients with MM; 2. QPCR, western blot (WB) and flow cytometry (FCM) to detect the level of macrophage polarization markers after constructing overexpression or knockdown of TRIM21; 3. WB and FCM to detect the sensitivity of MM cells to Bort after co-culture of MM cells and macrophage; 4. Transcriptome sequencing to identify TRIM21-mediated pathway changes in macrophage; 5. QPCR and WB to verify the effect of TRIM21 on macrophage polarization after treatment with pathway inhibitors. Results:1. Immunofluorescence analysis showed increased expression of TRIM21 in macrophage from relapsed and refractory MM (RRMM) patients compared to newly diagnosed multiple myeloma (NDMM) patients;2. QPCR, WB and FCM results showed that overexpression of TRIM21 in macrophage led to decreased polarization markers of M1 phenotype and increased the polarization markers of M2 phenotype. In contrast, knockdown of TRIM21 increased M1 polarization and decreased M2 polarization; 3. WB and FCM results demonstrated that co-culture of MM cells with TRIM21-overexpressing macrophages significantly enhanced MM cell tolerance to Bortezomib and decreased MM cell apoptosis; 4. Transcriptome sequencing revealed activation of the JAK-STAT3 pathway in macrophages after TRIM21 overexpression; 5. QPCR and WB results showed that the macrophage polarization effect induced by TRIM21 overexpression was reversed after treatment with the STAT3 inhibitor C188-9. Conclusions: Our study shows that TRIM21 is overexpressed in macrophages in patients with relapsed and refractory MM. In addition, TRIM21 induces M2 polarization by activating the JAK-STAT3 pathway, thereby enhancing the protective effect of macrophage on MM cells. These findings provide a new insight into the role of TRIM21 in MM drug resistance and lay an important theoretical foundation for targeting macrophages in MM. Key words: TRIM21, Macrophage, Bortezomib, JAK-STAT3

The selective activator protein-1 inhibitor T-5224 regulates the IRF4/MYC axis and exerts cooperative antimyeloma activity with bortezomib

The activating protein-1 (AP-1) transcription factors (TFs) have been associated with many different cancer types and are promising therapeutic targets in logical malignancies. However, the mechanisms of their role in multiple myeloma (MM) remain elusive. The present study determined and compared the mRNA and protein expression levels of the AP-1 family member JunB in CD138+ mononuclear cells from MM patients and healthy donors. Herein, we investigated the effect of T-5224, an inhibitor of JUN/AP-1, on MM. We found that the cytotoxicity of T-5224 toward myeloma is due to its ability to induce cell apoptosis, inhibit proliferation, and induce cell cycle arrest by increasing the levels of cleaved caspase3/7 and concomitantly inhibiting the IRF4/MYC axis. We also noticed that siJunB-mediated deletion of JunB/AP-1 enhanced MM cell apoptosis and affected cell proliferation. The software PROMO was used in the present study to predict the AP-1 TF that may bind the promoter region of IRF4. We confirmed the correlation between JunB/AP-1 and IRF4. Given that bortezomib (BTZ) facilitates IRF4 degradation in MM cells, we applied combination treatment of BTZ with T-5224. T-5224 and BTZ exerted synergistic effects, and T-5224 reversed the effect of BTZ on CD138+ primary resistance in MM cells, in part due to suppression of the IRF4/MYC axis. Our results suggest that targeting AP-1 TFs is a promising therapeutic strategy for MM. Additionally, targeting both AP-1 and IRF4 with T-5224 may be a synergistic therapeutic strategy for this clinically challenging subset of MM.

M6A methyltransferase METTL3 facilitates multiple myeloma cell growth through the m6A modification of BZW2.

N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) has been confirmed to be involved in multiple myeloma (MM) progression, and basic leucine zipper and W2 domains 2 (BZW2) is considered to be a regulator for MM development. However, whether METTL3 mediates MM progression by regulating BZW2 remains unclear. The messenger RNA (mRNA) and protein levels of METTL3 and BZW2 in MM specimens and cells were determined using quantitative real-time PCR and western blot analysis. Cell proliferation and apoptosis were assessed by cell counting kit 8 assay, 5-ethynyl-2'-deoxyuridine assay, colony formation assay, and flow cytometry. Methylated RNA immunoprecipitation-qPCR was used to detect the m6A modification level of BZW2. Xenograft tumor models were constructed to confirm the effect of METTL3 knockdown on MM tumor growth in vivo. Our results showed that BZW2 was upregulated in MM bone marrow specimens and cells. BZW2 downregulation reduced MM cell proliferation and promoted apoptosis, while its overexpression enhanced MM cell proliferation and inhibited apoptosis. METTL3 was highly expressed in MM bone marrow specimens, and its expression was positively correlated with BZW2 expression. BZW2 expression was positively regulated by METTL3. Mechanistically, METTL3 could upregulate BZW2 expression by modulating its m6A modification. Additionally, METTL3 accelerated MM cell proliferation and restrained apoptosis via increasing BZW2 expression. In vivo experiments showed that METTL3 knockdown reduced MM tumor growth by decreasing BZW2 expression. In conclusion, these data indicated that METTL3-mediated the m6A methylation of BZW2 to promote MM progression, suggesting a novel therapeutic target for MM.

Novel dual-targeting c-Myc inhibitor D347-2761 represses myeloma growth via blocking c-Myc/Max heterodimerization and disturbing its stability

BackgroundTranscription factor c-Myc plays a critical role in various physiological and pathological events. c-Myc gene rearrangement is closely associated with multiple myeloma (MM) progression and drug resistance. Thereby, targeting c-Myc is expected to be a useful therapeutic strategy for hematological disease, especially in MM.MethodsMolecular docking-based virtual screening and dual-luciferase reporter gene assay were used to identify novel c-Myc inhibitors. Cell viability and flow cytometry were performed for evaluating myeloma cytotoxicity. Western blot, immunofluorescence, immunoprecipitation, GST pull down and Electrophoretic Mobility Shift Assay were performed for protein expression and interaction between c-Myc and Max. c-Myc downstream targets were measured by Q-PCR and Chromatin immunoprecipitation methods. Animal experiments were used to detect myeloma xenograft and infiltration in vivo.ResultsWe successfully identified a novel c-Myc inhibitor D347-2761, which hindered the formation of c-Myc/Max heterodimer and disturbed c-Myc protein stability simultaneously. Compound D347-2761 dose-and time-dependently inhibited myeloma cell proliferation and induced apoptosis. Dual knockout Bak/Bax partially restored D347-2761-mediated cell death. Additionally, compound D347-2761 could, in combination with bortezomib (BTZ), enhance MM cell DNA damage and overcome BTZ drug resistance. Our in vivo studies also showed that compound D347-2761 repressed myeloma growth and distal infiltration by downregulating c-Myc expression. Mechanistically, novel dual-targeting c-Myc inhibitor D347-2761 promoted c-Myc protein degradation via stimulating c-Myc Thr58 phosphorylation levels, which ultimately led to transcriptional repression of CDK4 promoter activity.ConclusionsWe identified a novel dual-targeting c-Myc small molecular inhibitor D347-2761. And this study may provide a solid foundation for developing a novel therapeutic agent targeting c-Myc.CCo5k36iLrbCHdtx-yHunTVideo

Anti-Myeloma Effects of Panobinostat in Combination with Duvelisib

Introduction: Panobinostat (Secura Bio) is a non-selective histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. Duvelisib (Secura Bio) is an FDA-approved oral drug and a potent small molecule inhibitor of the delta (δ) and gamma (γ) isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. There is no data regarding in combination of an HDACi with duvelisib for multiple myeloma (MM) treatment. We evaluated the anti-myeloma effects of the panobinostat in combination with duvelisib.Methods: The human MM cell lines U266, RPMI 8226 and MM1s were obtained from ATCC. Primary MM tumor cells were isolated from MM patients' bone marrow (BM) aspirates and mononuclear cells (MCs) were isolated. The cells were seeded at 10 5 cells/well in 96-well plates and incubated for 24 h in the presence of vehicle, panobinostat or duvelisib alone, or the two drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. For the apoptosis assay, U266, RPMI 8226, MM1s, or primary MM tumor cells were cultured at 1 x 10 6 cells per well for 48 hours at 37°C in 5% CO2 with or without the addition of panobinostat or duvelisib. Next, the cells were washed twice with PBS, re-suspended in binding buffer, and stained with FITC-conjugated annexin V and fluorescent dye propidium iodide (PrI) following annexin V assay standard protocol (BioVision, USA). For each drug treatment, 1 x 10 5 gated events were recorded.Results and Discussion: The MTS cell proliferation assay showed that panobinostat alone inhibited cell proliferation of the MM cell lines U266 and MM1s but not RPMI8226. Panobinostat alone induced concentration-dependent inhibition of BMMCs. Panobinostat (20nM) in combination with duvelisib markedly increased inhibition of primary MM cell proliferation. Panobinostat alone induced MM cell apoptosis in all three MM cell lines. Panobinostat in combination with duvelisib enhanced MM cell apoptosis in MM1s but not U266 and RPMI 8226 cells. We further examined apoptosis of primary MM cell treated with panobinostat alone and in combination with duvelisib. The results showed panobinostat alone induced primary MM cell apoptosis and in combination with duvelisib markedly increased the induction of apoptosis. This study illustrates that the combination of panobinostat and duvelisib shows potent anti-MM effects in vitro and we are currently evaluating the anti-MM effects of this combination in vivo using our human MM xenograft models.*The study was partially funded by Secura Bio, Inc DisclosuresNo relevant conflicts of interest to declare.

OAB-021: BCMA-specific ADC MEDI2228 and Daratumumab induce synergistic myeloma cytotoxicity via enhanced IFN-driven innate immune responses and expression of CD38 and NKG2D ligands

<h3>Background</h3> Efforts are required to improve potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma (MM). <h3>Methods</h3> We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate MEDI2228 which may augment efficacy of these immunotherapies. RNA sequencing followed by gene set enrichment analysis showed that MEDI2228 significantly enriched IFN I-signaling and induced type I interferon (IFN I)-stimulated genes (ISGs) in MM cell lines. The most MEDI2228-enhanced IFN-driven genes include chemokines/cytokines and receptors (i.e., CXCL9/10, CCL4L1/2, CCL22, CCL1/3/4/5, CCL3L1/3, TNFSF9/10, CSF2 (GM-CSF), CCR7), RSAD2, CASP1, ISGs (i.e., XAF1, TRIMs, IFITs, ISG15, GBP2/3, OAS1/2/L, RNASEL, MX1/2, FAS), RUNX3, GZMB, IKBKE, IRF1/6/7/9, STAT1/2/4/6, MB21D1(CGAS), TMEM173 (STING), IFIT1/2/3/5, and SOCS1/2/3. <h3>Results</h3> Regardless of genetic heterogeneity and resistance to current anti-MM therapies, MEDI2228 induced dose- and time-dependent DNA damage-ATM/ATR-CHK1/2 pathways, activation of cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades, as well as increased CD38 expression. It overcame CD38 downregulation triggered by IL6 and bone marrow stromal cell culture supernatant (BMSC-sup), via activation of STAT1-IRF1 without phosphorylation of STAT3 in immunomodulatory drugs (IMiDs)- and bortezomib-resistant MM cell lines. In contrast, MEDI2228 did not change CD38 expression and survival in BCMA-negative NK effector cells. Significantly, MEDI2228 with anti-CD38 monoclonal antibody Daratumumab (Dara) synergistically induced NK-mediated lysis of MM cell lines and autologous resistant patient MM cells, even in the presence of BMSC-sup and BMSCs. In parallel, MEDI2228 increased membrane expression of NKG2D ligands (NKG2DLs), i.e., MICA/B and ULBPs, in all MM cells tested, including Dara-resistant patient cells, correlating with enhanced MM cell susceptibility to NK cell killing. Since MEDI2228, but not its MMAF-ADC homolog M3 even used at >1-log higher concentrations enhanced surface expression of CD38 and NKG2DLs on MM cells, the potent DDR-mediated immunomodulation triggered by MEDI2228 vs M3 is critical in rendering MM cells more susceptible to Dara-induced NK cell killing. Importantly, M2 still activated STAT1/IRF1 signaling to induce CD38 and MICA/B expression in MM tumors grown in mice. All MM1S tumor-bearing NSG mice reconsituted with human NK cells became tumor-free following a single low dose M2 with Dara treatment, with 100% host survival. <h3>Conclusions</h3> Taken together, our data showed that MEDI2228 restored MM sensitivity to CD38 targeting by Dara without depleting NK cells and potentiated immunogenic cell death of MM cells. These results therefore provide the mechanistic rationale for clinical evaluation of combination CD38- and BCMA-directed immunotherapies to further improve patient outcome in MM.

PRL-3 promotes a positive feedback loop between STAT1/2-induced gene expression and glycolysis in multiple myeloma.

Over 34 000 patients are diagnosed yearly with multiple myeloma (MM), which remains a fatal malignancy. Expression of the phosphatase PRL-3 is associated with poor prognosis in MM patients, and Vandsemb etal. have demonstrated that PRL-3 contributes to enhanced MM cell fitness through activation of a glycolysis-associated feedback loop. PRL-3 resulted in increased expression of signal transducer and activator of transcription 1 (STAT1) and 2 (STAT2) and increased glycolysis. Increased glucose metabolism in turn activated STAT1/2 and interferon 1-related genes. This discovery advances the MM field by providing a new potential treatment avenue. Comment on: https://doi.org/10.1111/febs.16058.

Upregulation of circ_0000142 promotes multiple myeloma progression by adsorbing miR-610 and upregulating AKT3 expression.

Circular RNAs (circRNAs) play an important regulatory role in a variety of malignancies. Nevertheless, the role of circ_0000142 in multiple myeloma (MM) and its regulatory mechanism remains largely unknown. Real-time polymerase chain reaction was employed to detect the expressions of circ_0000142 and miR-610 in MM tissues and cell lines. The expression of AKT3 and apoptosis-related proteins (Bcl-2, Bax) in MM cells was detected by western blot. The correlation between the expression level of circ_0000142 and the clinicopathological parameters of MM patients was analysed. Cell proliferation, apoptosis, migration and invasion were monitored by Cell Counting Kit 8 assay, flow cytometry analysis and Transwell assay, respectively. The dual-luciferase reporter gene assay and RNA immunoprecipitation assay were employed to verify the targeting relationship between circ_0000142 and miR-610. In this study, it was demonstrated that, circ_0000142 was highly expressed in MM patients, and its high expression level was significantly associated with increased International Staging System and Durie-Salmon stage. Overexpression of circ_0000142 enhanced MM cell proliferation, migration, invasion and suppressed cell apoptosis, while knocking down circ_0000142 had the opposite effects. Mechanistically, circ_0000142 functioned as a competitive endogenous RNA, directly targeting miR-610 and positively regulating AKT3 expression. In brief, circ_0000142 enhances the proliferation and metastasis of MM cells by modulating the miR-610/AKT3 axis.

&lt;p&gt;LINC00665 Promotes the Progression of Multiple Myeloma by Adsorbing miR-214-3p and Positively Regulating the Expression of PSMD10 and ASF1B&lt;/p&gt;

BackgroundAlthough assertion that long non-coding RNA (lncRNA) exerts crucial functions in the progression of multiple myeloma (MM) is well documented, few studies investigate function and underlying mechanism of long intergenic non-protein coding RNA 665 (LINC00665) in MM.Patients and MethodsA total of 25 MM patient samples and 15 healthy volunteer samples were collected, and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect the expressions of LINC00665. PSMD10 and ASF1B expressions were determined by qRT-PCR and Western blot assays. U266 cell and H929 cell were used in functional experiments. Besides, CCK-8 assay and flow cytometry analysis were utilized to determine cell proliferation and apoptosis. Bioinformatics analysis and dual-luciferase reporter assays were used to predict and verify the targeting relationships between LINC00665 and miR-214-3p, PSMD10 and miR-214-3p, as well as ASF1B and miR-214-3p. Moreover, the regulatory function of LINC00665 on the expression of PSMD10 and ASF1B was detected by Western blot.ResultsThe expression of LINC00665 was up-regulated in MM samples and cell lines. In vitro functional assays indicated that LINC00665 enhanced MM cell proliferation and inhibited its apoptosis. PSMD10 and ASF1B were identified as target genes of miR-214-3p. Additionally, LINC00665 negatively regulated miR-214-3p expression through sponging miR-214-3p and positively regulated PSMD10 and ASF1B.ConclusionLINC00665 can promote the expression of PSMD10 and ASF1B by inhibiting the expression of miR-214-3p, thus facilitating the proliferation and inhibiting apoptosis of MM cells.

The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

AbstractAngiogenic factors play a key role in multiple myeloma (MM) growth, relapse, and drug resistance. Here we show that malignant plasma cells (cell lines and patient-derived MM cells) express angiocrine factor EGF like-7 (EGFL7) mRNA and protein. MM cells both produced EGFL7 and expressed the functional EGFL7 receptor integrin β 3 (ITGB3), resulting in ITGB3 phosphorylation and focal adhesion kinase activation. Overexpression of ITGB3 or EGFL7 enhanced MM cell adhesion and proliferation. Intriguingly, ITGB3 overexpression upregulated the transcription factor Krüppel-like factor 2 (KLF2), which further enhanced EGFL7 transcription in MM cells, thereby establishing an EGFL7-ITGB3-KLF2-EGFL7 amplification loop that supports MM cell survival and proliferation. EGFL7 expression was found in certain plasma cells of patients with refractory MM and of patients at primary diagnosis. NOD.CB17-Prkdc<scid>/J mice transplanted with MM cells showed elevated human plasma EGFL7 levels. EGFL7 knockdown in patient-derived MM cells and treatment with neutralizing antibodies against EGFL7 inhibited MM cell growth in vitro and in vivo. We demonstrate that the standard-of-care MM drug bortezomib upregulates EGFL7, ITGB3, and KLF2 expression in MM cells. Inhibition of EGFL7 signaling in synergy with BTZ may provide a novel strategy for inhibiting MM cell proliferation.

NEK2 induces autophagy-mediated bortezomib resistance by stabilizing Beclin-1 in multiple myeloma.

NEK2 is associated with drug resistance in multiple cancers. Our previous studies indicated that high NEK2 confers inferior survival in multiple myeloma (MM); thus, a better understanding of the mechanisms by which NEK2 induces drug resistance in MM is required. In this study, we discovered that NEK2 enhances MM cell autophagy, and a combination of autophagy inhibitor chloroquine (CQ) and chemotherapeutic bortezomib (BTZ) significantly prevents NEK2‐induced drug resistance in MM cells. Interestingly, NEK2 was found to bind and stabilize Beclin‐1 protein but did not affect its mRNA expression and phosphorylation. Moreover, autophagy enhanced by NEK2 was significantly prevented by knockdown of Beclin‐1 in MM cells, suggesting that Beclin‐1 mediates NEK2‐induced autophagy. Further studies demonstrated that Beclin‐1 ubiquitination is decreased through NEK2 interaction with USP7. Importantly, knockdown of Beclin‐1 sensitized NEK2‐overexpressing MM cells to BTZ in vitro and in vivo. In conclusion, we identify a novel mechanism whereby autophagy is activated by the complex of NEK2/USP7/Beclin‐1 in MM cells. Targeting the autophagy signaling pathway may provide a promising therapeutic strategy to overcome NEK2‐induced drug resistance in MM.

MEDI2228, a Novel Bcma Antibody-PBD Conjugate, Sensitizes Human Multiple Myeloma Cells to NK Cell-Mediated Cytotoxicity and Upregulates CD38 Expression in MM Cells

MEDI2228, an antibody-drug conjugate (ADC) comprised of an anti-BCMA antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine dimer, is currently under clinical development for the treatment of human multiple myeloma (MM) (NCT03489525). MEDI2228 induces DNA damage responses (DDR) prior to apoptosis, as demonstrated by phosphorylation of ATM/ATR, CHK1/2, and gH2AX in MM cells regardless of p53 status and responsiveness to current MM therapies including bortezomib and IMiDs. Since activation of DDR alters expression of ligands for NKG2D receptors critical for NK-mediated immune surveillance, we here examined whether the ATM/ATR-CHK1/2 signaling cascades activated by MEDI2228 treatment would increase NKG2D ligands in MM cells. Using real-time quantitative RT-PCR and flow cytometry analysis, we found that treatment with MEDI2228 increased the expression of major histocompatibility complex (MHC) class I chain-related proteins A and B (MICA/B) in MM cell lines (n&gt;5) and CD138+ MM cells from patients with relapsed and refractory disease (n=4). In addition, expression of the MHC class I molecules/NKG2D ligands ULBP-1, -3, -2/5/6 increased following MEDI2228 treatment. Next, we evaluated NK cell-mediated lysis of MM target cells (n&gt;3) with or without pretreatment with MEDI2228 and found increased NK cell-mediated lysis of MEDI2228-pretreated vs control MM cells in an effector-target ratio-dependent manner. In parallel, we examined whether MEDI2228 stimulates STAT1- and IFN-related signaling pathways since they are activated by DDR and play a crucial role in innate and adaptive immunity. We found that MEDI2228 treatment significantly increases phosphorylation of STAT1 in H929 and its derived IMiD-resistant cells, and further augments expression of IFN-induced genes (IFITs), IFIT1, 2, 3, and 5, which have been shown to inhibit proliferation and promote apoptosis in cancer cells. Significantly, CD38 is upregulated by MEDI2228 treatment, with increased mRNA expression as well as membrane expression detected by flow cytometry in MM cell lines and MM cells from newly diagnosed and refractory patients (n=5). Consequently, MEDI2228-pretreated MM cells (n&gt;3) are more susceptible to NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) by daratumumab, which targets CD38. Taken together, our data show that MEDI2228-induced DDR primes MM cells to NK cell-mediated cytotoxicity by increasing expression of MICA/B in MM cells to enhance binding and activating NK cytolytic activity. Simultaneously, MEDI2228 induces IFN-stimulated genes, including CD38, resulting in enhanced MM cell lysis by daratumumab. These results indicate additional mechanisms of anti-MM activity for MEDI2228 and suggest that a combination of MEDI2228 and anti-CD38 mAbs may further improve outcome for MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau.

Anti-Bcma PBD MEDI2228 Combats Drug Resistance and Synergizes with Bortezomib and Inhibitors to DNA Damage Response in Multiple Myeloma

A novel anti-BCMA antibody drug conjugate (ADC) MEDI2228 preferentially binds to membrane-bound vs soluble BCMA (sBCMA) to effectively deliver the pyrrolobenzodiazepine (PBD) payload tesirine to multiple myeloma (MM) cells (Leukemia. 2019;33: 766). In preclinical models, this ADC targets both MM cells in bulk and CD19+CD138- patient MM progenitor cells. We here study the potency of MEDI2228 in MM cells in the bone marrow (BM) microenvironment and examine molecular mechanisms whereby MEDI2228 overcomes drug resistance. First, MEDI2228, more effectively than its microtubule-binding monomethyl auristatin-F (MMAF) ADC homolog, inhibits proliferation (&gt;1-2-log) and survival of all MM cell lines and MM cells from patients with multiple relapsed and refractory diseases, regardless of BCMA levels, p53 status, and the protection conferred by BM stromal cells and IL-6. Significantly, MM cells with lower BCMA expression and resistance to bortezomib or immunomodulatory drugs (IMiDs, i.e., lenalidomide, pomalidomide) are more susceptible to MEDI2228 vs its MMAF ADC homolog. MEDI2228, but not its MMAF ADC homolog, activates critical DNA damage responses (DDR) via phosphorylation of ATM/ATR kinases, checkpoint kinases (CHK)1/2, and H2AX, associated with induction of multiple DDR pathway-associated genes. Low doses of MEDI2228 and bortezomib (btz) synergistically induce apoptosis of drug-sensitive and -resistant MM cells, at least in part, through modulation of RAD51, a DNA damage and repair protein. Importantly, MEDI2228 further triggers the ATM/ATR-CHK1/2 signaling cascade, associated with increased gH2AX, p21, and apoptosis molecules in MM1S-xenografted tumors in mice. In vivo, a single sub-optimal dose of 0.4 mg/kg MEDI2228 induces superior anti-MM activity than btz, indicating that MEDI2228 is significantly more effective and selective than btz as single agent therapy in vivo. Furthermore, combined treatments with MEDI2228 and btz result in potent tumor depletion and significantly prolonged host survival via increased nuclear gH2AX-expressing microfoci, DNA damage-induced growth arrest and cell death. Significant tumor necrosis is observed earlier in mice receiving both drugs than either agent alone. At 177d, 15% mice in the combination treatment group remain alive and without any tumor. Importantly, no weight loss is noted in all groups, indicating a favorable safety profile of MEDI2228, alone or with btz, in vivo. Moreover, DDR checkpoint inhibitors, i.e., AZD0156 (ATMi), AZD6738 (ATRi), AZD1775 (WEE1i), synergize with MEDI2228 to enhance MM cell cytotoxicity (combination index &lt; 1). This study therefore further supports clinical development of MEDI2228 (NCT03489525) as an important next-generation immunotherapy to improve outcome of MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Amgen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board.

GFI1-Dependent SGPP1 Repression Promotes Growth and Survival of Myeloma Cells

Multiple myeloma (MM) remains incurable for the vast majority of patients due to emergence of drug resistant clones and mutations inducing drug resistant relapses. This is despite the fact that new therapies have greatly improved progression-free and overall survival for patients with standard risk myeloma. We recently found that the transcriptional repressor GFI1 is increased in bone marrow stromal cells of MM patients (MM-BMSC) where it causes prolonged suppression of osteoblast differentiation, and in CD138+ cells from MM patients, where GFI1 levels significantly correlate with disease progression. We also found that GFI1 overexpression (o/e) enhances MM cell growth and partially confers resistance to proteasome inhibitors in vitro as well as enhances tumor growth and osteoclastogenesis in vivo. Although the mechanisms responsible for these GFI1 effects in p53wt MM cells were p53-dependent, we found that GFI1 is also essential for MM cell survival regardless of their p53 status. The p53-independent mechanisms responsible for Gfi1 effects on MM cells growth and survival of are unknown. Sphingolipids are bioactive lipids that can control MM cell growth and survival. The balance between the levels of Sphingosine-1-phosphate (S1P) and its metabolic precursors ceramide (Cer) and sphingosine (SPH) form a rheostat that determines whether a cell proliferates or dies. We hypothesized that GFI1 represses SGPP1, the enzyme responsible for degrading S1P via salvage and recycling of sphingosine into long-chain ceramides. This repression changes the intracellular sphingolipid profile (Cer/S1P/SPH ratio) to maintain c-Myc upregulation in a protein phosphatase 2 (PP2A)-dependent manner, thus promoting growth and survival of MM cells. To test this hypothesis we measured S1P, SPH and Cer levels by mass spectrometry (LC-MS/MS). LC-MS/MS evaluation showed that bone marrow plasma of MM patients has significant higher levels of S1P when compared to normal donors. Moreover, intracellular S1P levels of MM.1S GFI1 o/e cells were also significantly higher as compared to those of MM.1S empty vector controls. Knock-down (KD) of Gfi1 in MM.1S cells strikingly increased SGPP1 and decreased SphK1 (the enzyme which catalyzes S1P production) mRNA levels, while GFI1 o/e cells had the opposite effect. We found that CD138+ cells isolated from MM patients expressed elevated levels of SphK1 mRNA compared to MGUS patients, and that SphK1 protein levels directly correlate with GFI1 levels in MM patient CD138+ cells and cell lines (r= 0.527). We also detected an indirect correlation (r= -0.961) between GFI1 and SGPP1 mRNA levels in five different MM cell lines. These results indicate a GFI1-dependent imbalance of the enzymes regulating S1P production. Further, KD GFI1 and SphK1 inhibition (5 μM SK1I) had a profound inhibitory effect on c-Myc protein levels and induced caspase 3 activation as detected by Western blotting, while GFI1 o/e cells had significant higher levels of c-Myc and were more resistant to SK1I treatment. Exogenous ceramide (10 μM Cer 16:0) treatment or SphK1 inhibition (5 μM SK1I), both treatments known to trigger intracellular ceramide production, significantly inhibited MM cell viability (measured by AlamarBlue), regardless of their p53 status (MM1.S p53 +/+ and KMS-11 p53 -/-). This inhibition of MM viability was GFI1-dependent, as GFI1 o/e cells were significantly more resistant to ceramide-induced cell death, which was PP2A dependent, as PP2A inhibition with okadaic acid (OA) restored it. MM.1S cells with KD of GFI1 exhibited significantly higher PP2A activity then control cells, supporting our observation that c-Myc modulation by GFI1 is PP2A-dependent. c-Myc protein levels were significantly decreased in MM.1S control cells treated with ceramide and rescued by OA pre-treatment; thus mimicking the effects of changing GFI1 levels and I2PP2A (the PP2A endogenous inhibitor) and confirms that PP2A mediates the effects of GFI1 on c-Myc. Taken together, our results show that GFI1 acts as a key regulator of MM growth and survival, at least partially through modulation of SGPP1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees.

The Gfi1-SphK1 Axis Regulates the Growth and Survival of Myeloma Cells

In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown.Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio).We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1.Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. DisclosuresRoodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Growth factor independence 1 expression in myeloma cells enhances their growth, survival, and osteoclastogenesis

BackgroundIn spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown.MethodsHuman primary CD138+ and BMSC were purified from normal donors and MM patients’ bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo.ResultsWe found that Gfi1 expression is increased in patient’s MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth.ConclusionsThese results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.

CRISPR knock out of programmed cell death protein 1 enhances anti-tumor activity of cytotoxic T lymphocytes.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that functions to attenuate T cell activation. In this study, we knocked out (KO) PD-1 in cytotoxic T lymphocytes (CTLs) using CRISPR-Cas9 system to evaluate its effect on the anti-tumor activity of the CTLs against multiple myeloma (MM). Results show that PD-1 KO CTLs facilitate apoptosis and caspase activation of the co-cultured MM cells and enhanced MM cell death by 36% compared with the control. PD-1 KO also increased TNF-α and IFN-γ secretion of the CTLs by 2.4 and 1.9-fold respectively. The effectiveness of PD-1 KO in enhancing anti-tumor activity of the CTLs was verified in vivo using mouse xenograft model. The xenografted mice treated with PD-1 KO CTLs demonstrated repressed MM tumor growth and prolonged survival compared with the control group. We conclude that CRISPR-Cas9 is an efficient system to knock out PD-1 from CTLs and PD-1 KO could significantly enhance the anti-tumor activity of CTLs.

Biomarker Driven Phase II Clinical Trial of Trametinib in Relapsed/Refractory Multiple Myeloma with Sequential Addition of the AKT Inhibitor, GSK2141795 at Time of Disease Progression to Overcome Treatment Failure: A Trial of the Princess Margaret Phase II Consortium

Activating mutations of the MAPK pathway are reported in over half of myeloma tumors. Experience with MEK inhibitors in solid tumors suggest that although tumors harboring BRAF or RAS mutations are more likely to respond, response rates are low and duration of responses short. Potential explanations include the activation of alternative signaling pathways and in particular PI3K/AKT signaling. Studies of multiple myeloma (MM) tumors suggest that AKT activation is independent of oncogenic RAS and that combined inhibition of RAS and AKT enhances MM cell death. Based on these observations, we initiated a clinical trial to evaluate the activity of trametinib (TMTB) in MM patients (pts) with or without RAS/RAF mutations, as a single-agent and in combination with AKT inhibition for pts who fail to respond to TMTB alone.Methods: Pts were independently recruited into biomarker positive (K/NRAS or BRAF mutated) or biomarker negative (K/NRAS, BRAF wild type) groups. All pts received TMTB, 2 mg/day on a 28 day cycle. In pts who developed progressive disease (PD) or achieved less than a partial response (PR) after 4 cycles of TMTB monotherapy, GSK2141795 (pan-AKT inhibitor) was added. TMTB combined with GSK2141795 was dosed at 1.5 mg/50 mg taken daily. The M-protein at the time of adding GSK2141795 was considered the new baseline for response assessment to the combination. The main objectives were to evaluate overall response rates (ORR; IMWG) to TMTB in the 2 groups and determine the clinical benefit of adding GSK2141795 to TMTB.Results: At the data cutoff, 25 pts were enrolled: 12 in the mutated (MUT) and 13 in the wild type (WT) groups. Median age and prior lines of therapy were 65 years (range 42-76) and 4 (range 4-8) for the MUT group and 64 years (range 51-81) and 4 (range 2-8) for the WT group. 96%, 100%, 60% and 96% of pts had received prior proteasome inhibitor (PI), immunomodulatory drugs (IMiDs), pomalidomide or PI+IMiD therapy, respectively. The most common (>30%) adverse events (AEs) possibly related to study drugs were thrombocytopenia (40%) and diarrhea (32%) for TMTB monotherapy and nausea (72%), rash (63%), thrombocytopenia (36%) and anorexia (36%) with the addition of GSK2141795. The most frequent grade 3-4 AEs (>20%) were thrombocytopenia (24%) for TMTB monotherapy and thrombocytopenia (45%), anemia (45%), lymphopenia (36%), hyponatremia (27%), and neutropenia (27%) in combination with GSK2141795. One death occurred due to gastrointestinal bleed unrelated to study drugs.Of 24 pts assessable for response, confirmed ORR was 8% (1 PR) for the MUT group (N=12), Further, 1 pt had an unconfirmed minimal response (MR) and 4 pts had stable disease (SD). For the WT group (N=12), we observed 2 MR (1unconfirmed) and 3 SD. For clinical benefit rate (CBR) of 17% in both groups. With the addition of GSK2141795 to TMTB (N=11), the ORR was 27% (3 PR, 1 unconfirmed) and 1 pt had an MR (CBR=36%). The median PFS is estimated to be 3.2 (range 0.9-3.8) and 1.8 (range 0.9-NR) months (p=0.91), for the MUT and WT groups, respectively.Correlative studies are ongoing and are designed to identify predictors of response and resistance to TMTB. These include, assessment of pre- and post-treatment expression of phospho-ERK1/2, serial monitoring of clonal dynamics in bone marrow and cell-free DNA and pharmacogenomic studies. Analyses will be presented.Conclusions: The MEK1/2 inhibitor, TMTB demonstrates clinical activity in MM pts with RAS mutated tumors. In addition, disease control was observed for pts with WT RAS/RAF tumors. However, single agent activity in heavily pre-treated pts is modest. The addition of GSK2141795 to block the alternative signaling pathway improved the ORR to 27% supporting further exploration of this treatment strategy for MM. DisclosuresTrudel:Celgene: Consultancy, Equity Ownership, Honoraria; Novartis: Consultancy, Honoraria; Glaxo Smith Kline: Honoraria, Research Funding; Oncoethix: Research Funding; BMS: Honoraria; Amgen: Honoraria. Bahlis:Amgen: Consultancy, Honoraria; BMS: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau. Venner:Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Research Funding; J+J: Research Funding; Janssen: Honoraria. Hay:Amgen: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Kite Pharmaceuticals: Research Funding.