Growth Inhibition Of Myeloma Cells Research Articles

Ethanol extract of Eclipta prostrata induces multiple myeloma ferroptosis via Keap1/Nrf2/HO-1 axis

BackgroundMultiple myeloma (MM) is an incurable hematological malignancy with limited therapeutic efficacy. Eclipta prostrata is a traditional Chinese medicinal plant reported to possess antitumor properties. However, the effects of E. prostrata in MM have not been explored. PurposeThe aim of this study was to define the mechanism of the ethanol extract of E. prostrata (EEEP) in treating MM and identify its major components. MethodsThe pro-ferroptotic effects of EEEP on cell death, cell proliferation, iron accumulation, lipid peroxidation, and mitochondrial morphology were determined in RPMI-8226 and U266 cells. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), and 4-hydroxynonenal (4HNE) were detected using western blotting during EEEP-mediated ferroptosis regulation. The RPMI-8226 and U266 xenograft mouse models were used to explore the in vivo anticancer effects of EEEP. Finally, high performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry system (UPLC-Q/TOF-MS) were used to identify the major constituents of EEEP. ResultsEEEP inhibited MM cell growth and induced cell death in vitro and in vivo. By promoting malondialdehyde and Fe2+ accumulation, lipid peroxidation, and GSH suppression, EEEP triggers ferroptosis in MM. Mechanistically, EEEP regulates the Keap1/Nrf2/HO-1 axis and stimulates ferroptosis. EEEP-induced lipid peroxidation and malondialdehyde accumulation were blocked by the Nrf2 activator NK-252. In addition, HPLC and UPLC-Q/TOF-MS analysis elucidated the main components of EEEP, including demethylwedelolactone, wedelolactone, chlorogenic acid and apigenin, which may play important roles in the anti-tumor function of EEEP. ConclusionIn summary, EEEP exerts its anti-MM function by inducing MM cell death and inhibiting tumor growth in mice. We also showed that EEEP can induce lipid peroxidation and accumulation of ferrous irons in MM cells both in vivo and in vitro, leading to ferroptosis. In addition, this anti-tumor function may be achieved by the EEEP activation of Keap1/Nrf2/HO-1 axis. This is the first study to reveal that EEEP exerts anti-MM activity through the Keap1/Nrf2/HO-1-dependent ferroptosis regulatory axis, making it a promising candidate for MM treatment.

Development of a T cell-redirecting bispecific antibody targeting B-cell maturation antigen for the suppression of multiple myeloma cell growth.

BackgroundMultiple myeloma (MM) is the second most common hematological malignancy. It has emerged as one of the next possible hematological diseases amenable to immunotherapy. B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily, is highly expressed in MM cells and is one target with the most potential for developing MM-targeting immunotherapy. Other than the FDA-approved BCMA-targeting CAR T-cell therapy, such as Abecma and CARVYKTI, T cell-engaging multi-specific antibody is another promising therapeutic modality for BCMA-targeting MM treatment. We develop a T-cell redirecting BCMA-targeting bispecific antibody (bsAb) and evaluate its anti-MM activity.MethodsWe first generated several clones of mouse anti-human BCMA monoclonal antibodies using DNA immunization. One of the anti-BCMA antibodies was then used to design and produce a T cell-redirecting BCMA × CD3 bsAb in CHO cells. Finally, we examined the effect of the bsAb on MM cell growth both in vitro and in vivo.ResultsThe BCMA × CD3 bsAb was designed in a FabscFv format and produced in CHO cells with good yield and purity. Moreover, the bsAb can trigger robust T cell proliferation and activation and induce efficient T cell-mediated MM cell killing in vitro. Using a MM xenograft mouse model, we demonstrate that the bsAb can effectively suppress MM cell growth in vivo.ConclusionsOur results suggest that the BCMA × CD3 bsAb in the FabscFv format can efficiently inhibit MM cell growth and have promising potential to be developed into a therapeutic antibody drug for the treatment of MM.

The Novel Dual Topoisomerase Inhibitor P8-D6 Shows Anti-myeloma Activity In Vitro and In Vivo.

P8-D6 is a novel dual inhibitor of human topoisomerase I (TOP1) and II (TOP2) with broad pro-apoptotic antitumor activity. NCI-60 screening revealed markedly improved cytotoxicity of P8-D6 against solid and leukemia cell lines compared with other single and dual topoisomerase inhibitors, for example, irinotecan, doxorubicin, or pyrazoloacridine. In this study, we investigated the capacity of P8-D6 to inhibit myeloma cell growth in vitro and in vivo Growth inhibition assays demonstrated significant anti-myeloma effects against different myeloma cell lines with IC50 values in the low nanomolar range. Freshly isolated plasma cells of patients with multiple myeloma were killed by P8-D6 with similar doses. P8-D6 activated caspase 3/7 and induced significant apoptosis of myeloma cells. Supportive effects of bone marrow stromal cells on IL6-dependent INA-6 myeloma cells were abrogated by P8-D6 and apoptosis occurred in a time- and dose-dependent manner. Of note, healthy donor peripheral blood mononuclear cells and human umbilical vein endothelial cells were not affected at concentrations toxic for malignant plasma cells. Treatment of myeloma xenografts in immunodeficient SCID/beige mice by intravenous and, notably, also oral application of P8-D6 markedly inhibited tumor growths, and significantly prolonged survival of tumor-bearing mice.

Combined Nanoparticle Delivery of PARP and DNA-PK Inhibition for Multiple Myeloma

Double strand DNA damage repair is increased in multiple myeloma (MM) and may be responsible for clonal evolution and resistance to chemotherapy. The enzyme poly [ADP-ribose] polymerase-1 (PARP-1) is integral to the DNA damage response and is involved in single strand repair as well as homologous recombination. DNA-dependent protein kinase (DNA-PK) is central to non-homologous end joining. Inhibition of PARP or DNA-PK have both been shown to lead to cytotoxicity in myeloma cells. In addition, we have shown that combined inhibition leads to synergistic cytotoxicity as well as enhancing the effect of alkylator chemotherapy. In order to efficiently deliver combined PARP/DNA-PK inhibition on the individual cell level, we developed a unique combined nanoparticle comprising olaparib (PARP inhibitor) and a prodrug of PI-103 (DNA-PK inhibition).We first synthesized an SN2 lipase-labile phosphatidylcholine prodrug of PI-103. The chemical structure of the prodrug was confirmed by 1H-nuclear magnetic resonance, low resolution mass spectrometry and high resolution mass spectrometry. The prodrug is able to self-assemble into a phospholipid bilayer, which formed the outer layer of the nanoparticle. Olaparib becomes incorporated into the core of the colloidal suspension due to its hydrophobicity. For the particle to be active against MM cells it would fuse with the cell membrane and then be internalized. Subsequently the outer prodrug layer would be enzymatically cleaved and activated and olaparib would be released from the inner core. The hydrodynamic diameter of developed nanoparticles was found to be <150nm with -20mV electrophoretic potential values indicating their colloidal stability. These particles were able to incorporate very high concentration of drug molecules (> 85%) and exhibited an enzymatic release of ~30% for PI-103 and ~50% for olaparib at 24 hours under low pH conditions of 4.0.We subsequently tested the ability of the combination nanoparticle to induce cytotoxicity in RPMI-8226 myeloma cells by treating cells with increasing doses in liquid culture. In a standard proliferative assay (3[H] thymidine uptake), the combination nanoparticle efficiently inhibited proliferation with an IC50 of 1.3microM. As a comparison, we analyzed the cytotoxicity of particles containing olaparib alone (nano-olaparib), PI-103 alone (nano-PI-103) or control nanoparticles without either drug. Control nanoparticles did not exert significant cytotoxicity. At a physiologic dose of 5microM, combination particles resulted in 96.7% inhibition in proliferation in comparison to nano-olaparib (55.3%, p=0.03) and nano-PI-103 (71.5%, p=0.03). In addition, at 5microM concentrations, combination particles resulted in significantly greater inhibition of proliferation than when cells were treated with both nano-olaparib particles and nano-PI-103 particles (84.9%, p=0.03). Treatment with combination nanoparticles resulted in a dose dependent increase in G2M arrest and rapid induction of apoptosis within 3 hours with a 5microM dose leading to a 15% absolute increase in Annexin+ cells. Immunofluorescent staining for γH2AX after 24-hour treatment with combination particles also showed a marked dose dependent increase, confirming that cytotoxicity was a result of double strand DNA damage. In order to establish that combined PARP and DNA-PK inhibition was safe in normal hematopoietic cells, we treated normal CD34+ cells with increasing doses of combination particles up to 5microM in a standard colony assay in methylcellulose medium supplemented with cytokines. Colony numbers were unaffected by particle treatment. Finally we safely treated an RPMI-8226 NOD/SCID/gamma null xenograft model of myeloma with 1.5g/kg of combination nanoparticles. Final results including a comparison with non-nanoparticle standard drugs will be presented at the meeting.We present the proof of principle that our novel nanoparticle combined PARP/ DNA-PK inhibition is feasible and potently inhibits myeloma cell growth in-vitro but does not affect normal CD34+ cells. In the future, it is possible that this drug delivery platform could be utilized to efficiently deliver other synergistic drug combinations. DisclosuresPatel:Celgene: Consultancy, Honoraria.

Abstract 6559: Novel anti-myeloma potential of CAPE analogs: Downregulation of IKZF1-IRF4-MYC axis

Abstract Background: Multiple myeloma (MM) is the second most common hematological malignancy and an incurable bone marrow cancer so far. The hallmark of myeloma is chromosomal translocations that transform plasma cells into malignant myeloma cells. 50-75% of myeloma patients exhibit translocations at the immunoglobulin heavy chain (IgH) locus juxtaposing oncogenes from partner chromosome under the control of strong 3' IgH enhancers. Overexpression of oncogenes such as Cyclin D1, Cyclin D3, interferon regulatory factor (IRF4) etc. occurs, depending on the translocating partner locus. We have earlier reported that elevated expression of octameric binding protein-2 (OCT2), a key transcription factor in IgH translocations as a poor prognostic factor and shown its association with reduced survival in MM patients. IRF4 is another indispensable transcription factor for plasma cell differentiation, and its juxtaposition with IgH locus leads to IRF4 overexpression mediated tumorigenesis. Both OCT2 and IRF4 are regulated by the transcription factor, nuclear factor kappa B (NF-κB). Current chemotherapeutics exhibit several side effects, and face the challenge of chemoresistance, thus warranting the need for novel therapeutics for myeloma. Caffeic acid phenethyl ester (CAPE) is an active principle of propolis from honeybee hives, with potent NF-κB inhibitory activity. Hence, we synthesized and evaluated the structure-activity relationship of twenty-one CAPE analogs for their anti-myeloma potential. Methods: Human myeloma cell lines, lenalidomide, CAPE and its analogs were used for the study. Myeloma cell growth inhibition by CAPE analogs was determined by PrestoBlue cell viability assays. Effect of CAPE analogs on IRF4, OCT2, CyclinD3, caspase-3, Ikaros (IKZF1), MYC levels were evaluated by qRT-PCR and/or immunoblotting methods; apoptosis induction detected by Annexin V-PI flow cytometry assays. Results: Cyclohexylethyl and two phenpropyl ester analogs exhibited high myeloma cell growth inhibition in comparison with CAPE and lenalidomide. IRF4 was significantly downregulated both at mRNA and protein levels by the phenpropyl ester analog that demonstrated a remarkable increase in cleaved caspase-3 levels and apoptotic cell number. For the first time, we have demonstrated the ability of CAPE analogs to decrease OCT2, IKZF1, IRF4 and MYC levels in myeloma cells. Downregulation of the IKZF1-IRF4-MYC axis by CAPE and its key analogs revealed their novel anti-myeloma mechanism of action. Furthermore, the lead analogs showed no adverse cytotoxic effect on normal human mononuclear or naïve B-cells, also exhibited appropriate in silico pharmacokinetic properties and drug-likeness. Conclusion: Thus, our findings suggest the promising application of CAPE analogs to target Ikaros/IRF4 addiction, the so-called Achilles heel of myeloma, thereby leading to better therapeutic outcomes. Citation Format: Alli Murugesan, Gregoire Lasalle-Claux, Lauren Hogan, Elise Vaillancourt, Ayyoub Selka, Katie Luiker, Andy Hong, MinJi Kim, Mohamed Touaibia, Tony Reiman. Novel anti-myeloma potential of CAPE analogs: Downregulation of IKZF1-IRF4-MYC axis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6559.

Multidimensional Single Cell Analysis Shows Increased T/NK Cell Subsets in Both Blood and Bone Marrow of Iberdomide (CC-220) Treated Relapsed/Refractory Multiple Myeloma Patients

Background: Iberdomide (IBER), a novel cereblon (CRBN) E3 ligase modulatory compound (CELMoD), is being evaluated in a phase 1/2 clinical trial for treatment (tx) of RRMM, as a monotherapy, in combination with low-dose dexamethasone (DEX), or in combination with DEX and daratumumab, bortezomib, or carfilzomib (CC-220-MM-001; NCT02773030). IBER modulates the CRBN E3 ligase, inducing ubiquitination and proteasome-dependent degradation of Ikaros, Aiolos, and ZFP91, resulting in inhibition of myeloma-cell growth and immunomodulation. Methods: As of June 28, 2019, 95 patients (pts) had received IBER as monotherapy or in combination with DEX at doses ranging from 0.3 to 1.3 mg taken once daily (QD) on Days 1-21 of 28-day cycles (21/28). Longitudinal immune profiling by peripheral blood (PB) flow cytometry was completed at Cycle (C) 1 Day (D) 1 and C2D15 in 90 pts dosed up to 1.3 mg. T cell and NK cell proliferation was assessed by Ki67 staining and T cell activation was assessed by HLA-DR expression. T cell differentiation was assessed using markers for CD45RA, CD45RO, and CCR7. To assess immune changes in the bone marrow (BM) microenvironment, viably frozen CD138- cells from BM aspirates were obtained from pts at screening and C2D15 (10 and 6 samples available respectively; dosed from 0.3 - 0.9 mg). Samples were analyzed using both single-cell RNA sequencing (scRNA-seq) and mass cytometry by time-of-flight (CyTOF) with a novel panel designed to identify B cells, T and NK cell (sub)populations, monocytes, dendritic cells, MDSCs and Tregs. An R based workflow using the CATALYST (Crowell et al. 2019) and Seurat package (Stuart et al. Cell 2019) was used to analyze CyTOF and scRNA-seq data, respectively. Results: PB immune profiling of pts showed increases in total and proliferating NK cells following IBER tx across all doses (median increase = 35.5% and 74.2%, respectively). A dose-dependent increase in proliferating, activated and effector memory CD4+ (median increase = 92.3%, 45.5%, 51.7%, respectively, across all doses) and CD8+ (median increase = 116.0%, 13.9%, 45.8%, respectively, across all doses) T cells was also observed. To understand if these changes were concurrent with changes in the tumor microenvironment, a novel CyTOF panel was produced to analyze BM samples. Analogous to the PB findings, total BM NK cells increased (6.05% cells at screening vs. 9.91% at C2D15). CD16+ NK cells, known for their cytolytic activity, increased most prominently (35.9% of NK cells to 42.4%) vs. CD16- NK cells (64.1% of NK cells to 57.6%). Higher median CD16, CD56 and HLA-DR surface expression at C2D15 was also observed vs screening, demonstrating increased abundance and differentiation of NK cells. Similar to observations in the T cell compartment of PB, decreases in naïve CD4+ (24.4% to 9.09% of CD4+ cells) and CD8+ (6.42% to 2.14% of CD8+ cells) T cells and increases in effector memory CD4+ (35.8% to 40.1%) and CD8+ (26.7% to 39.5%) T cells were observed in BM. Transcriptomic data in BM showed increased expression of genes associated with cytolytic/cytotoxic activity (NKG7, GZMB, CCL3/4) and the interferon response pathway (IFNG, TNF, IFIT2/3) in CD8+ T cells, further supporting a shift towards activation in BM T cells with IBER tx. Conversely, we observed decreased expression of genes associated with a resting state (KLF2, ITGB7, CD52, S100A4) on CD4+ T cells in BM. Consistent with data from PB and of other immunomodulatory drugs, we also observed an increase in CD4+ regulatory T cells upon tx (0.72% to 5.06% of CD4+ T cells) in BM, however, the consequence of this observation in terms of response remains unclear. Conclusions: Pharmacodynamic profiling of immune subsets in PB and BM of RRMM pts receiving IBER showed consistent changes in NK cells and T cell subsets, suggesting increased proliferation and a more activated phenotype. Ongoing analyses will address whether such patterns of immune cell responses are more pronounced at higher doses of IBER and how they may correlate with clinical outcome. Disclosures Amatangelo: Celgene Corporation: Employment, Equity Ownership. Gooding:Celgene: Research Funding. Oppermann:Bayer Pharmaceuticals: Research Funding; GSK: Research Funding; Celgene: Research Funding. Pierceall:Celgene: Employment. Thakurta:Celgene: Employment, Equity Ownership. Parekh:Karyopharm Inc.: Research Funding; Foundation Medicine Inc.: Consultancy; Celgene Corporation: Research Funding.

Novel ZBP1-IRF3 Dependency in Multiple Myeloma Mediated By IRF3-Driven Regulation of Cell Cycle Genes

ZBP1 is an inducible nucleic acid (NA) sensor that is activated when pathogen NA bind to its Zα and Zβ domains. ZBP1 is required for TBK1-dependent phosphorylation of the transcription factor IRF3 (pIRF3) followed by its direct activation of type I interferon genes. However, the role, if any, of ZBP1 in tumour biology is not known. By searching for genes selectively expressed in multiple myeloma (MM) we identified ZBP1 mRNA expressed in 29 MM cell lines (MMCL) but not in &gt;1000 other cancer cell lines (CCLE dataset); ZBP1 was expressed in all 766 patient myeloma PC (CoMMpass dataset) but not in normal blood cells (Blueprint) or 53 healthy tissues (GTex). We confirmed expression of ZBP1 mRNA and/or protein in MMCL, primary human and murine germinal centre B (GCB) and plasma cells (PC) as well as in myeloma PC. By inducing T cell-dependent humoral immune responses after ip alum-NP-KLH immunisation, we explored the role of selective and constitutive expression of Zbp1 in GCB to PC transition. We found no differences in the frequency of splenic GCB cells and PC between control WT and Zbp1-/- mice and in GCB cell frequency between immunised WT and Zbp1-/- mice. However, compared to WT, the increase in PC frequency in immunised Zbp1-/- mice was 50% lower (n=10/group, p&lt;0.0001) commensurate with a 40% (n=6/group, p&lt;0.01), lower increase in NP-KLH-specific IgG but not IgM levels in Zbp1-/- mice. These findings suggest that although Zbp1 is not required for GCB cell and PC development it is required for optimal, T cell-dependent humoral immune responses. To explore the function of ZBP1 in MM we depleted by 2 lentiviral shRNAs either isoform 1 (contains both Zα and Zβ domains; shRNA1) or both isoform 1 and isoform 2 (latter lacks Zα domain; shRNA2). Both shRNAs were toxic to all 5 MMCL tested suggesting that isoform 1 but not isoform 2 is essential for myeloma cell survival. This effect was specific because survival of K562 cells, which lack expression of ZBP1, was not affected by either shRNA and exogenous ZBP1 cDNA rescued cell death of ZBP1-depleted myeloma cells. Dox-induced ZBP1 depletion was toxic to MMCL in vitro and significantly inhibited myeloma cell growth in a subcutaneous NSG model of the MMCL H929 and MM.1S. Together, these findings reveal a novel myeloma cell-specific ZBP1 dependency. Transcriptome analysis of ZBP1-depleted H929 and MM.1S cells showed amongst the significantly downregulated genes enrichment for the cell cycle control and DNA repair pathways consistent with a critical role of ZBP1 in promoting myeloma cell proliferation. Flow-cytometric analysis of ZBP1-depleted MMCL as well as of patient-derived myeloma PC revealed cell cycle arrest at the G0/1 phase and increasing apoptosis. Exploring potential links with IRF3, we first observed that unlike in non-malignant cells, IRF3 was constitutively phosphorylated in MMCL. Using protein-co-immunoprecipitation we found that endogenous ZBP1 interacts with IRF3 and TBK1 while upon co-transfection with different ZBP1 deletion mutants, ZBP1-IRF3 interaction required primarily the ZBP1, RHIM domain-containing, C-terminus. Further, while in ZBP1-depleted myeloma cells total IRF3 and TBK1 levels were not altered, pIRF3 and pTBK1 levels decreased thus showing a post-translational dependency of constitutive pIRF3 and pTBK1 on ZBP1. Finally, pharmacological inhibition of TBK1 resulted in decrease of pIRF3 without affecting total IRF3. Importantly, shRNA-mediated IRF3 depletion resulted in cell cycle arrest and death of MMCL. By integrating histone mark and in-house IRF3 ChiP-seq with transcriptome of IRF3-depleted MM.1S cells we identified 770 down- and 330 up-regulated genes predicted to be directly regulated by IRF3. Pathway enrichment analysis confirmed cell cycle as the most highly regulated by IRF3. Notably, we observed no direct or indirect regulation of the interferon genes (e.g., IFNA1, IFNB1) by IRF3. As well as the IRF3 motif, IRF3 cistrome analysis revealed significant enrichment for the distinct IRF4 motif. Integration of the IRF3/IRF4 cistromes identified &gt;80% IRF3 binding regions are co-occupied by IRF4 and co-regulation of cell cycle genes. Further we validated IRF3-IRF4 interaction at the IRF4 super-enhancer by ChIP-re-ChIP. These data show a novel dependency in MM comprising constitutive activation of the ZBP1-IRF3 pathway and regulation of cell cycle and proliferation by IRF3 thus providing opportunities for therapeutic targeting. Disclosures Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.

PF559 IBERDOMIDE (CC‐220) IS PHARMACODYNAMICALLY ACTIVE AND HAS DOSE‐DEPENDENT IMMUNOSTIMULATORY ACTIVITY IN RELAPSED/REFRACTORY MULTIPLE MYELOMA PATIENTS IRRESPECTIVE OF PRIOR IMID DRUG TREATMENT

Background:Iberdomide (IBER) is a novel cereblon (CRBN) E3 ligase modulatory compound (CELMoD) that is being investigated as monotherapy, in combination with dexamethasone (DEX), and in combination with DEX and daratumumab or bortezomib in a phase 1b/2a dose‐escalation study in relapsed/refractory multiple myeloma (RRMM). IBER modulates CRBN, which induces ubiquitination of Aiolos, Ikaros and ZFP91, increases their proteasome‐dependent degradation, and thereby inhibits myeloma cell growth. IBER binds more potently to CRBN and is more efficient at degrading Aiolos and Ikaros than the immunomodulatory agents (IMiDs) lenalidomide (LEN) and pomalidomide (POM) and it can overcome IMiD drug resistance in pre‐clinical models.Aims:To determine if IBER is pharmacodynamically (PD) active in multiple myeloma patients refractory to POM and/or LEN, and to establish pharmacokinetic (PK)/PD and PD/dose relationships.Methods:As of Jan 11, 2019, 83 patients received IBER as monotherapy or in combination with DEX; 61 (74%) were LEN refractory and 47 (57%) were POM refractory. Longitudinal immunohistochemistry (IHC) data on CRBN, Aiolos, Ikaros and ZFP91 expression was obtained from bone marrow samples from 14 patients dosed with 0.3–0.9 mg IBER daily ± DEX. PD analysis of immune activity by TruCulture® (n = 45) and by peripheral blood flow cytometry (n = 60) was completed on a subset of patients dosed up to 1.0 mg IBER ± DEX. A population PK approach was used to estimate exposure (area under the concentration curve over the 24 hr dosing interval [AUCτ]) on 59 patients dosed up to 1.0 mg IBER ± DEX.Results:IBER AUCτ increased in a dose‐proportional manner in RRMM patients over the dose range 0.3–1.0 mg (22.4–65.31 ng∗hr/mL, respectively). In patient biopsies analyzed to date, CRBN expression was observed in all tumors by IHC at baseline and did not significantly change while on‐treatment. Furthermore, no significant decrease in baseline CRBN expression was observed in patients refractory to POM. In MM cells, significant decreases in Aiolos (median 37% decrease, P &lt; 0.001), Ikaros (median 12% decrease, P = 0.006), and ZFP91 (median 47% decrease, P &lt; 0.001) protein expression were observed on‐treatment across all dose levels; these decreases were unrelated to levels of CRBN expression at screening or prior LEN or POM refractoriness. In TruCulture® samples, dose‐dependent increases were observed in IL‐2 and IFN‐γ, with median increases of 78% and 66%, respectively, 5 hours post 0.9 mg dose of IBER. A dose‐ and exposure‐dependent decrease in B‐cells was detected (median 74% decrease at cycle 2 day 15, IBER 0.9 mg). These observations are consistent with the proposed IBER mechanism of action. Immunoprofiling showed significant increases in total and proliferating NK cells in RRMM patients treated with IBER, and a dose‐dependent increase in proliferating, activated, and effector memory T‐cells. These trends were consistent in RRMM patients treated with IBER monotherapy and in combination with DEX.Summary/Conclusion:These translational analyses from IBER treated RRMM patients indicate it is pharmacodynamically active at the lowest dose (0.3 mg) and exhibits dose‐ and exposure‐dependent immunostimulatory activity, supporting further dose‐escalation beyond 1.0 mg, which is currently ongoing. These results also suggest that prior refractoriness to LEN or POM is not associated with significant loss of CRBN or IBER PD activity, supporting continued investigation of IBER in RRMM patients refractory to IMiD drugs.

The Novel Bromodomain and Extraterminal Domain Inhibitor INCB054329 Induces Vulnerabilities in Myeloma Cells That Inform Rational Combination Strategies.

Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials. We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations. In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK-STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone. Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

Knockdown of long non-coding RNA PCAT-1 inhibits myeloma cell growth and drug resistance via p38 and JNK MAPK pathways.

Objective: Both previous and recent literature showed long non-coding RNAs (lncRNAs) were crucial participants in multiple myeloma (MM) evolution. However, the dynamic regulation and mechanism of lncRNAs in MM progression was not fully understood. This study will explore the expression and effects of prostate cancer-associated ncRNA transcript 1 (PCAT-1) in MM.Materials and Methods: The expression level of PCAT-1 was examined using quantitative real-time PCR in patients with newly diagnosed MM and cell lines. The potential biological effects and molecular mechanisms of PCAT-1 in MM were evaluated using a series of soft agar colony formation assay, CCK-8 assay, cell cycle and apoptosis assay by flow cytometry, protein chip arrays, western blot analysis, immunohistochemistry and nude subcutaneous tumorigenesis model.Results: High expression of PCAT-1 was observed in patients with newly diagnosed MM and cell lines. Over-expressed PCAT-1 enhanced cell division and inhibited apoptosis both in cultured cells and in animal model. Meanwhile, silenced PCAT-1 exerted the opposite function. Additionally, PCAT-1 knockdown sensitized MM cells to bortezomib (Bort). Inhibitor of PCAT-1 combination with Bort exhibited a more effective inhibitory effect on MM cells compared with negative control or Bort alone. Further mechanism exploration via protein chips, Go and KEGG pathway analysis along with immunoblot analysis revealed that PCAT-1 facilitated cell growth and drug resistance via the p38 and JNK MAPK pathways.Conclusion: This study identified a novel lncRNA-associated mechanism underlying MM carcinogenesis, and provided clinicians with a promising therapeutic target in MM.

Abstract 2498: Over-expression of dual-specificity phosphatase 4 (DUSP4) in multiple myeloma

Abstract Super-enhancers are unique genomic domains with dense binding of transcription factors (TFs)/cofactors that drive expression of genes defining cell identity. They also contribute to over-expression of oncogenes. The abundance of multiple TFs and coactivators at the super-enhancers favors their cooperative binding and synergistic gene activation. Thus, super-enhancers are more sensitive to perturbation of TF levels than typical enhancers, providing a therapeutic window for preferential targeting oncogenic expression in cancer cells. Recently, a list of super-enhancer associated genes (SEAG) was identified in a multiple myeloma (MM) cell line MM1S and inhibition of transcriptional coactivator BRD4 by JQ1 selectively repressed MYC oncogene expression through reduction of BRD4 binding at the associated super-enhancer. Significances of SEAG other than MYC in myeloma cells remains unclear. By unsupervised hierarchical clustering analysis of expression of 682 identified SEAG in 74 MM, 28 relapsed MM and 15 normal plasma cells (NPC) (GSE6477), distinct expression pattern of SEAG was associated with MM as compared to NPC, indicating their importance in MM. Among the upregulated genes, DUSP4 (Dual Specificity Phosphatase 4), an inducible phosphatase of MAPK (Mitogen-Activated Protein Kinases), was significantly overexpressed in MM and further upregulated in the relapsed MM. High expression was associated with inferior patient outcomes. The overexpression was confirmed in our patient cohort which includes CD138-immunosorted myeloma cells from 30 untreated and 3 relapsed MM patients, 6 smoldering MM patients and 5 MGUS patients, 5 sorted normal plasma cells (NPC) and 7 MM cell lines. Using the cutoff for over-expression as higher than the mean level of NPC plus 2X Standard Deviation, 10 out of 30 MM and all the 3 relapsed samples showed significant DUSP4 over-expression. Functional significance of DUSP4 in MM was explored by siRNA knockdown and the knockdown inhibited myeloma cell growth, implying an oncogenic role of DUSP4 in MM. To study the significances of super-enhancer in regulation of DUSP4 expression, JQ1 was used and inhibition of BRD4 by JQ1 in MM1S, L363 and OPM2 repressed both DUSP4 expression and MM cell growth. By ChIP-PCR, we demonstrated BRD4 specifically bound onto the proximal region of DUSP4 promoter and the binding was blocked by JQ1. Taken together, the oncogenic role of DUSP4 as SEAG in MM was revealed and the preferential targeting SEAG by BRD4 inhibitor could provide insights into further developments of effective therapeutic approaches for MM patients. Citation Format: Tian Xia, Kin-Mang Lau, Chi Keung Cheng, Nelson CN Chan, Margaret H. L. Ng. Over-expression of dual-specificity phosphatase 4 (DUSP4) in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2498.

Metformin induces autophagy and G0/G1 phase cell cycle arrest in myeloma by targeting the AMPK/mTORC1 and mTORC2 pathways

BackgroundMetformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-tumor effects in many cancers, including multiple myeloma (MM); however, the underlying molecular mechanisms have not been clearly elucidated.MethodsThe anti-myeloma effects of metformin were evaluated using human MM cell lines (RPMI8226 and U266) in vitro and in vivo NOD-SCID murine xenograft MM model. Cell viability was assessed with CCK8 and cell proliferation was measured by EdU incorporation assay. Cell cycle distribution and apoptosis were examined by flow cytometry. Transmission electron microscopy was used to visualized autophagosomes. Activation of AMPK and inhibition of mTORC1/C2 pathways was assessed by Western blot analysis. RPMI8226 cells and U266 cell lines with AMPK knockdown were generated by transfection with small interfering RNA targeting the AMPK-α1 and α2 subunits using Lipofectamine 2000 reagent.ResultsMetformin effectively inhibited the proliferation of MM cell lines, an effect that was associated with the induction of autophagy and G0/G1 cell cycle arrest, but not apoptosis. Metformin activated AMPK and repressed both mTORC1 and mTORC2 signaling pathways in myeloma cells as well as downstream molecular signaling pathways, such as p-4EBP1 and p-AKT. AMPK activation resulted in direct phosphorylation and activation of tuberous sclerosis complex 2 (TSC2), leading to inhibition of the mammalian target of rapamycin (mTOR). In addition, metformin inhibited myeloma cell growth in an AMPK-dependent manner. The xenograft mouse model further confirmed that metformin inhibited tumor growth by upregulation of AMPK and downregulation of mTOR.ConclusionsMetformin inhibits the proliferation of myeloma cells by inducing autophagy and cell-cycle arrest. Our results suggest that the molecular mechanism involves dual repression of mTORC1 and mTORC2 pathways via AMPK activation. Our study provides a theoretical basis for the development of novel strategies for the treatment of MM using metformin as an already approved and safe drug.

Abstract 5121: PEG(E)-TC11, a novel polyethylene glycol-linked phthalimide derivative, inhibited high-risk MM cell growth in vivo and in vitro via cell cycle G2/M arrest in a CRBN-independent manner

Abstract Multiple myeloma (MM) is one of the hematological malignancy that is characterized by proliferation of malignant plasma cells. Recent advance in the treatment of MM using newly developed drugs, prognosis of the MM patients have been significantly improved. For example, immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have been developed for treatment of MM. However, IMiDs have only limited effects against MM patients with high risk chromosomal abnormalities such as t(4;14) and del17p (high-risk MM). In 2010, it was reported that IMiDs directly bind to cereblon (CRBN), a component of ubiquitin ligase 3 complex, and induced teratogenicity as well as anti-tumor effects. We have previously reported that a novel phthalimide derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11) induced apoptosis against high-risk MM cells in vivo and in vitro, and inhibited differentiation of osteoclasts. We also reported that TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1), but did not bind to CRBN. However, TC11 was not well dissolved in water with only 0.02 mg/mL solubility. Therefore, TC11 showed poor absorption into blood and limited anti-tumor activity when it was intraperitoneally administered in tumor-bearing mice. To resolve these problems, we synthesized PEG(E)-TC11, in which TC11 is linked to polyethylene glycol through an ester bond, and consequently enhanced water solubility of PEG(E)-TC11 to 88.9 mg/mL. PEG(E)-TC11 revealed as potent growth inhibitory effect on high-risk MM cells as TC11 in vitro. In pharmacokinetic study, PEG-modification of TC11 improved the peak blood concentration (Cmax) from 2.6 to 24.4 μM and extended elimination half-life (t1/2) from 1.4 to 2.2 hr when 186 μM/kg of these compounds were intraperitoneally injected. More importantly, these pharmacokinetic improvement led to more potent growth inhibition of MM cells in vivo than TC11. We also explored mechanisms of anti-myeloma effect of PEG(E)-TC11 and found that PEG(E)-TC11 induced apoptosis via G2/M cell cycle arrest. However, unlike IMiDs family, BIACORE assay revealed that PEG(E)-TC11 didn’t directly bind to CRBN, indicating that growth inhibitory effect of PEG(E)-TC11 against MM cells was independent of binding to CRBN. In conclusion, PEGylation of TC11 significantly increased water solubility, resulted in potentiated anti-myeloma activity in vivo. Furthermore, PEG(E)-TC11 inhibited cell growth via G2/M arrest in a CRBN-independent manner. Thus, PEG(E)-TC11 is considered as a candidate compound for overcoming high-risk MM. Citation Format: Shuji Aida, Daiju Ichikawa, Kazuki Iida, Masashi Hozumi, Misa Nakamura, Ryo Uozaki, Nahoko Hashimoto, Mikio Okayama, Yuko Yonemura, Noriko Tabata, Taketo Yamada, Maiko Matsushita, Takeshi Sugai, Hiroshi Yanagawa, Yutaka Hattori. PEG(E)-TC11, a novel polyethylene glycol-linked phthalimide derivative, inhibited high-risk MM cell growth in vivo and in vitro via cell cycle G2/M arrest in a CRBN-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5121. doi:10.1158/1538-7445.AM2017-5121

TRIP13 impairs mitotic checkpoint surveillance and is associated with poor prognosis in multiple myeloma.

AAA-ATPase TRIP13 is one of the chromosome instability gene recently established in multiple myeloma (MM), the second most common and incurable hematological malignancy. However, the specific function of TRIP13 in MM is largely unknown. Using sequential gene expression profiling, we demonstrated that high TRIP13 expression levels were positively correlated with progression, disease relapse, and poor prognosis in MM patients. Overexpressing human TRIP13 in myeloma cells prompted cell growth and drug resistance, and overexpressing murine TRIP13, which shares 93% sequence identity with human TRIP13, led to colony formation of NIH/3T3 fibroblasts in vitro and tumor formation in vivo. Meanwhile, the knockdown of TRIP13 inhibited myeloma cell growth, induced cell apoptosis, and reduced tumor burden in xenograft MM mice. Mechanistically, we observed that the overexpression of TRIP13 abrogated the spindle checkpoint and induced proteasome-mediated degradation of MAD2 primarily through the Akt pathway. Thus, our results demonstrate that TRIP13 may serve as a biomarker for MM disease development and prognosis, making it a potential target for future therapies.

Effect of levamisole on expression of CD138 and interleukin-6 in human multiple myeloma cell lines.

Multiple myeloma (MM) is a B-cell malignancy accounting for 0.8% of all cancer deaths globally. This malignancy is characterized by lytic bone disease renal insufficiency, anemia, hypercalcemia, and immunodeficiency. The myeloma cells have enhanced expression of CD138. CD138 is a transmembrane heparin sulfate glycoprotein expressed on different types of adherent and nonadherent cells.CD138 is used as a standard marker for identification of tumor cells. Despite introduction of many therapeutic agents, the management of multiple myeloma (MM) remains a challenge and search for new therapeutic agents is in progress. In this study, we attempted to evaluate the effect of an alkaline phosphatase inhibitor, levamisole on expression of CD138, and level of interleukin-6 (IL-6) in human MM cell lines RPMI 8226 and U266 B1. U266B1 and RPMI 8226 cell lines were obtained from the National Centre for Cell Sciences, Pune. Alkaline phosphatase assay, Interleukin-6 assay and CD138 expression on myeloma cells by flow cytometry were investigated when the cells were exposed to Levamisole. Levamisole-mediated growth inhibition of myeloma cells in vitro is associated with a loss of CD138 and increased IL-6 secretion. The increased secretion of IL-6 by myeloma cells could be an attempt to protect themselves from apoptosis. Levamisole inhibited CD138 expression and affected the levels of IL-6 in a dose-dependent manner. The results of the present study add new dimension to levamisole's mode of action as inhibitor of CD138 and IL-6 and as an antiapoptotic agent.

Inhibition of the Epigenetic Modifier EZH2 Upregulates Cell Cycle Control Genes to Inhibit Myeloma Cell Growth and Overcome High-Risk Disease Features

IntroductionHigh expression of the H3K27 histone methyltransferase EZH2 mRNA in myeloma (MM) patient samples is associated with molecular features of high risk disease, including increased proliferation, and adverse outcomes (1). Mutations or deletions in the H3K27 demethylase KDM6A are associated with similar findings (2) and would be expected to have the same epigenetic effect, increasing H3K27me3 levels, a mark associated with repression of gene expression. We, therefore, sought to identify the role EZH2 plays in controlling myeloma cell proliferation.MethodsA panel of MM cell lines and primary patient samples (CD138 selected from bone marrow with consent) representing a variety of different MM molecular subgroups were used. Cell viability (WST-1), cell cycle (PI) and apoptosis (AnnexinV/PI, Caspase-Glo 3/7) assays were performed. Affymetrix gene expression arrays followed by validation with RT-PCR were used to identify patterns of gene expression change with EZH2i. Western blotting confirmed changes at the protein level and Chip-PCR was performed using a validated antibody and isotype control to identify H3K27me3 changes at the relevant gene promotors. Affymetrix gene expression data for 1213 patients enrolled in the Total Therapy studies were used to investigate the relevance of our findings in myeloma patient samples.ResultsWe confirmed a reduction in viability following EZH2i using two chemically distinct, specific small molecule inhibitors (EPZ005687 and UNC1999) and the negative control compound UNC2400. There was a reduction in viability in 6/8 cell lines and 5/6 patient samples. Response to inhibition was not related to molecular subgroup or the presence of high-risk molecular features including del17p. Global levels of H3K27me3 measured by Western blot were reduced in all cell lines regardless of response to EZH2i. In responding cell lines EZH2i induced cell cycle arrest at G1/S followed by induction of apoptosis.Gene expression arrays performed using mRNA from KMS11 and KMM1 cell lines highlighted a change in expression of cell cycle control genes associated with EZH2i. This finding was validated using qRT-PCR, which demonstrated upregulation of the cyclin dependent kinase inhibitors CDKN2B, CDKN1A or both. These findings were confirmed at the protein level by Western blotting.Chip-PCR experiment using cell lysates from KMS11 cells following incubation with EZH2i over 6 days identified changes in H3K27me3 at the promoter and transcriptional start site (PROM/TSS) regions of the CDKN2B and CDKN1A genes. The most specific changes occurred at the CDKN1A PROM/TSS, which were more heavily marked with H3K27me3 at baseline compared to a region approx. 5KB upstream.Given these results, which suggest that CDKN1A expression may be controlled by changes in H3K27me3, we explored the effect of CDKN1A mRNA expression in our patient datasets. We found the expression of EZH2 and CDKN1A to be inversely correlated (R=-0.170, p<0.0001) and that low expression of CDKN1A was associated with a significantly shorter progression free and overall survival (p<0.001).In order to confirm whether these gene expression changes could be used as a potential biomarker of response we looked at our panel of cell lines with variable responses to EZH2i. We identified a consistent increase in expression of CDKN1A only in responding cell lines suggesting it could be used as a biomarker of efficacy in the clinic.ConclusionsThese data support the hypothesis that CDKN1A expression is suppressed by increased H3K27me3, due to high expression of EZH2 and that this can be reversed with pharmacological EZH2 inhibition leading to a reduction in proliferation of myeloma cells. We provide data which supports the investigation of EZH2i in clinical trials of myeloma patients, which has the potential to be an effective therapeutic strategy even for those with high-risk disease, for whom current treatment approaches are ineffective.Pawlyn et al, EZH2 Overexpression in Myeloma Patients Shortens Survival and in-vitro Data Supports a Potential New Targeted Treatment Strategy. AACR and IMW abstracts, 2015Pawlyn et al, The Spectrum and Clinical Impact of Epigenetic Modifier Mutations in Myeloma. Clinical Cancer Research, 2016 DisclosuresPawlyn:Celgene: Consultancy, Honoraria, Other: Travel Support; Takeda Oncology: Consultancy. Kaiser:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; BMS: Consultancy, Other: Travel Support; Takeda: Consultancy, Other: Travel Support; Chugai: Consultancy. Jones:Celgene: Honoraria, Research Funding. Jackson:Amgen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau. Bergsagel:Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Bristol Meyers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

The Trifunctional Antibody (tri-31C2) Targeting CD38 and CD3 Has a Stronger Anti-Myeloma Effect Than the Chimeric Anti-CD38 Monoclonal Antibody (ch-31C2)

[Introduction]The application of trifunctional, bispecific antibodies directed against CD38, a transmembrane glycoprotein, and CD3, a T-cell antigen, may be an effective therapeutic strategy for multiple myeloma. Hence, in this study we constructed and evaluated such antibody. The trifunctional antibody with an Fc region has the potential to engage natural killer cells and macrophages. This antibody efficiently recruited effector T cells to CD38-expressing myeloma cells and induced antibody-dependent cell-mediated cytotoxicity (ADCC).[Methods]The recombinant protein consisted of the extracellular domain of the human CD38 (aa 43-300), which was fused to the N-terminus of the Fc region of the human IgG1 and expressed in CHO-K1. These CD38-expressing cells were used to immunize mice. Monocytic cells from the spleen of the immunized mice were fused with a fusion partner, X63-Ag8.653 mouse myeloma cell line, using polyethylene glycol-mediated cell fusion. This was followed by the selection of a hybridoma using the method of Kinebuchi et al. One of the monoclonal antibodies (31C2, IgG1 κ) was confirmed to recognize CD38. The VH and VL genes of the hybridoma cells were cloned with the synthesized primers, using previously reported polymerase chain reaction (PCR) methods. The expression vectors of the chimeric and trifunctional antibodies, ch-31C2 and tri-31C2, respectively, were constructed from the cloned genes. Following this, we constructed the chimeric anti-CD38 monoclonal antibody (ch-31C2) as well as the trifunctional antibody (tri-31C2) which binds CD38 and CD3 and evaluated their anti-myeloma activities.[Results]We assessed the potential of tri-31C2 to inhibit CD38-positive cell growth. CD38-positive RPMI 8226 and CD38-negative Colo 320 cells were treated with tri-31C2, ch-31C2, at concentrations of 0.1 and 1 µg/mL respectively; human polyclonal IgG was used as the control. These cells were subsequently co-cultured with PBMCs (E/T ratio 5). The results revealed that 1 but not 0.1 µg/mL of the ch-31C2 inhibited the cell viability by approximately 80-90% compared to the control. In contrast, both 0.1 and 1 µg/mL of the tri-31C2 inhibited myeloma cell growth to 30% viability compared to the control.Co-culturing the CD38-positive RPMI 8226 myeloma cells and PBMCs with 0.1 and 1.0 µg/mL of tri-31C2 triggered significant secretion of TNFα, INFγ, and IL-2; however, the same concentrations of ch-31C2 did not increase cytokine secretion. Furthermore, there was an insignificant increase in secretion of these cytokines when CD38-negative Colo 320 cells were co-cultured with ch-31c2 or tri-31C2 Next, we examined the antitumor effects of the tri-31C2 and ch-31C2 antibodies in a mouse xenograft model of multiple myeloma. They were both found to significantly inhibit tumor formation compared to the control.[Conclusion]We have successfully generated the chimeric anti-CD38 monoclonal antibody (ch-31C2) as well as the trifunctional antibody (tri-31C2) which binds CD38 and CD3. Our results suggest that tri-31C2 had a stronger anti-myeloma effect compared to ch-31C2. DisclosuresNo relevant conflicts of interest to declare.

Cyclin-Dependent Kinase 4/6 Inhibitor Abemaciclib Exerts Dose-Dependent Cytostatic and Cytocidal Effects on Multiple Myeloma Cells Via Autophagy

Background:Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of abnormal plasma cells in the bone marrow. D-type cyclins (CCNDs), an important family of cell cycle regulators, are thought to be implicated in multiple myeloma (MM) development because CCNDs are commonly expressed in myeloma cells. CCND is known to positively regulate the cell cycle from G1 to S-phase initiation by binding to cyclin-dependent kinase (CDK) 4/6, resulting in potentiation of myeloma cell growth. These findings suggest a possible role for CDK4/6-targeting therapy in MM, yet the details remain incompletely understood. In this regard, we investigated the biological activity of abemaciclib, a potent, highly selective CDK4/6 inhibitor, in myeloma cell lines, to elucidate the mechanisms underlying the involvement of the CCND-CDK4/6 complex in cell cycle regulation and survival.Methods:The effects of abemaciclib on myeloma cells were investigated using three myeloma cell lines, KMS12-PE (CCND1-positive and CCND2-negative), RPMI8226 (CCND1-negative and CCND2-positive), and IM-9 (both CCND1- and CCND2-positive). Cell growth was assessed by trypan blue exclusion assay. Cell cycle analysis was performed using propidium iodide (PI) and apoptosis was measured using annexin V/PI staining via flow cytometry. Cell cycle regulated proteins, including p21 and p27, and phosphorylated proteins, including STAT1, STAT3, ERK, JNK, p38, and AKT, were evaluated using a phospho-flow method. Autophagy was assessed using CYTO-ID via flow cytometry. PARP cleavage was investigated via western blotting. Clarithromycin, an antibiotic agent belonging to the macrolide class, was used as an autophagy inhibitor.Results:Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all the cell lines evaluated, with significant differences seen at a concentration of 320 nM. Annexin V/PI staining revealed that 1 μM abemaciclib showed little or no effect on apoptosis, but 3.2 μM abemaciclib induced apparent myeloma cell apoptosis, with an increase in both the early and late apoptotic fractions. Therefore, 1 and 3.2 μM of abemaciclib were used in subsequent experiments for the assessment of cell growth and apoptosis, respectively. Cell cycle analyses revealed that 1 μM abemaciclib increased the fraction of cells in G0/G1 phase and decreased the fraction in S-G2/M phase. Furthermore, this effect was associated with the upregulation of p21 and p27 in the evaluated myeloma cells. PARP cleavage was observed in KMS12-PE cells treated with 3.2 μM abemaciclib, but not 1 μM, suggesting a close connection between the degree of PARP cleavage and apoptosis in myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner. However, no apparent inhibitory effect on the autophagy-related phosphorylated proteins STAT1 (Y701), STAT3 (Y705), ERK (T202/Y204), JNK (T183/Y185), p38 (T180/Y182), or AKT (Y315) was observed in myeloma cells treated with 3.2 μM abemaciclib. To investigate the role of abemaciclib-induced autophagy on myeloma cell apoptosis, we further assessed the apoptotic effect of 3.2 μM abemaciclib or 50 μg/mL clarithromycin, alone or in combination. Clarithromycin did not induce apoptosis of myeloma cells. Importantly, clarithromycin treatment in combination with abemaciclib attenuated the apoptotic effect of abemaciclib.Discussion & Conclusions: Although the underlying mechanisms conferring the level of CCND expression are known to differ greatly (e.g., CCND translocation, hyperdiploidy, or activation of upstream pathways of CCND transcription), the results of the current study indicate that the CCND-CDK4/6 complex is closely involved in myeloma cell growth and survival regardless of the CCND family member present. In addition, we demonstrate that abemaciclib exerts multiple effects, such as myeloma cell apoptosis, via the PARP pathway or autophagy, as well as cell cycle regulation. Because abemaciclib in combination with clarithromycin inhibits myeloma cell apoptosis, the autophagy induced by abemaciclib is considered to have a critical role in the induction of apoptosis, so-called "autophagic cell death." These results provide novel insights into a possible therapeutic approach using abemaciclib to target CDK4/6 in patients with MM, and offer new possibilities for combination therapy with CDK4/6 inhibitors and autophagy regulators. DisclosuresIriyama:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Hatta:Novartis Pharma: Honoraria.

Abstract 3791: TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents

Abstract Background: Multiple myeloma and its associated bone disease are generally incurable. Hence, better therapies are needed, ideally targeting biomolecules implicated in the aberrant biology of the disease. Overexpression of transient receptor potential cation channel TRPV6, a highly selective calcium channel has been observed in breast, colon, thyroid, ovary and prostate cancer tissues, and in several tumour cell lines. TRPV6 expression has also been seen in osteoclasts, however its role in bone metabolism remains unclear. The reciprocal interaction between osteoclasts and myeloma cells is pivotal to the generation of bone lesions that characterize myeloma. While myeloma cells secrete factors promoting osteoclast activity, osteoclasts in turn are known to induce myeloma cell growth and survival. The TRPV6 peptide antagonist SOR-C13 is currently in phase I clinical trials as an anti-cancer therapy for advanced cancers. We investigated the expression and potential therapeutic significance of TRPV6 in human osteoclasts and myeloma cells using shrew venom derived peptide antagonists. Methods: High affinity TRPV6 peptide antagonists SOR-C13 and SOR-C27 derived from the C-terminus of venom from the short-tailed shrew, Blarina brevicauda were used. Human primary osteoclasts were generated in vitro from human bone marrow (BM) aspirates; characterized by Hoechst-phalloidin staining, Tartrate resistant acid phosphatase (TRAP) staining, TRAP enzyme activity and Cathepsin K expression. CD138 positive myeloma cells were isolated from patient bone marrow specimens by EasySep immunomagnetic separation, or examined in tissue microarrays of patient BM core biopsies. TRPV6 expression in primary human osteoclasts, myeloma cell lines and myeloma patient BM microarray was checked by qPCR, immunohistochemistry or immunoblotting. Anti-resorptive potential of SOR peptides using human osteoclasts was evaluated in Osteoassay plates that mimic bone, and myeloma cell growth inhibition was determined by prestoblue cell viability assays. Results: We found strong expression of TRPV6 protein in human myeloma cells and osteoclasts by immunohistochemical staining on myeloma patient bone marrow sections. Human osteoclasts generated in vitro and CD138 positive myeloma patient bone marrow plasma cells were found to express TRPV6. We saw dose-dependent inhibition of osteoclast activity in vitro by SOR-C13 and SOR-C27, including markedly reduced osteoclast formation, decreased TRAP activity and reduced osteoassay surface resorption. TRPV6 peptide antagonists were also found to inhibit the growth of human myeloma cell lines U266 and KMM-1. Conclusion: Anti-myeloma and anti-osteoclast activity of human TRPV6 antagonist peptides was seen in the current study. Taken together, our findings suggest a novel therapeutic approach for multiple myeloma involving TRPV6 inhibition to target both myeloma cells and osteoclasts. Citation Format: Alli Murugesan, Philippe Tremblay, Ming Han, Bithika Ray, Tyler Lutes, Tony Reiman. TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3791.

Impact of pomalidomide therapy in multiple myeloma: a recent survey

Pomalidomide (Pomalyst®) is a synthetic compound derived by modifying the chemical structure of thalidomide to improve its potency and reduce its side effects and third drug in the class of immunomodulatory drugs. Pomalidomide is under global development with Celgene Corporation, was approved by the U.S. Food and Drug Administration on February 8, 2013 to treat patients with relapsed and refractory multiple myeloma (MM) who have received at least two prior therapies including bortezomib and lenalidomide. In October 2009, it has found orphan designation for the treatment of relapsed and refractory MM by the EMA, and on August 2013, marketing authorization has issued in Europe by gaining a positive response. It inhibits myeloma cell growth and angiogenesis directly. Pomalidomide is the latest myeloma cell growth inhibitor to be approved in both USA and EU. The predominant side effects are thrombocytopenia, neuropathy, and deep vein thrombosis. Pomalidomide is also being investigated in patients with amyloidosis, prostate cancer, small cell lung cancer, pancreatic cancer, graft-versus-host disease, and Waldenstrom’s macroglobulinemia. This article reviews the available information on pomalidomide with respect to its clinical pharmacology, mechanism of action, pharmacokinetics, pharmacodynamics, metabolism, pre-clinical studies, and clinical trials.