ALBERT

Description: Myeloma (MM) cells grow and expand almost exclusively in the bone marrow while creating a cellular microenvironment suitable for MM cell growth and survival (MM niche). In pursuing the molecular mechanisms whereby MM cells gain drug resistance in the “MM niche”, we have found that the serine/threonine kinase Pim-2 is constitutively over-expressed in MM cells, and further up-regulated by co-cultures with bone marrow stromal cells (BMSCs) as well as osteoclasts (Leukemia, 2011), and that Pim-2 is an important therapeutic target in MM for the progression of MM tumor and bone disease (Leukemia, 2014). The ABC transporter BCRP is preferentially expressed in drug resistant MM cells as well as in MM progenitors or stem cells. BCRP has been demonstrated to be phosphorylated by Pim kinases to trigger its dimerization and function; Pim inhibition may suppress the BCRP function to sensitize BCRP-expressing MM cells to chemotherapeutic agents. In the present study we therefore explored whether Pim inhibition is able to target and impair BCRP-expressing drug-resistant MM cells and MM progenitors. We analyzed an ABC transporter activity in BCRP-expressing RPMI8226 and KMS11 cells by intracellular accumulation and retention of BCRP substrates with auto-fluorescence emission, mitoxantrone and doxorubicin, in flow cytometry. Treatment with Pim inhibitors, SMI-16a or SMI-4a, increased the incorporation of these drugs into the MM cells and enhanced their subsequent intracellular retention after 6-hour incubation without these drugs, although BCRP expression on their surface was only marginally affected by the Pim inhibition. Interestingly, acidic conditions up-regulated Pim-2 expression while reducing the accumulation and retention of these drugs in BCRP-expressing RPMI8226 and KMS11 cells. However, the Pim inhibitors efficaciously restored the drug accumulation and retention reduced by extracellular acidification, and enhanced the cytotoxic activity of the BCRP substrate doxorubicin against RPMI8226 cells rather preferentially in acidic conditions. Furthermore, the Pim inhibition minimized the sizes of “side populations”, highly drug-resistant fractions with enhanced BCRP activity, and the ability of colony formation in RPMI8226 and KMS11 cells, which was more marked in acidic conditions. We previously demonstrated the in vivo effects of the Pim inhibitors in human INA-6 cell-bearing SCID-rab MM models and syngeneic mouse MM models with an intra-tibial inoculation of 5TGM1 MM cells (Leukemia, 2014). To further examine the acid-tropism of anti-tumorigenic activity of Pim inhibition, we pretreated murine 5TGM1 MM cells in vitro with or without SMI16a at pH6.8 for 24 hours, and transplanted to the tibiae in mice the same numbers of viable MM cells remaining in each treatment group. Treatment with SMI16a at pH6.8 almost completely abrogated in vivo tumorigenic capacity of 5TGM1 cells, while MM cells without the treatment rapidly grew and expanded in and outside of the tibiae, suggesting targeting clonogenic MM cells by Pim inhibition preferentially in acidic conditions. Taken together, Pim-2 may become an important therapeutic target of drug-resistant BCRP-expressing MM cells and their progenitors which appear to gain more drug resistance in acidic bone lesions. Combinatory treatment with Pim inhibitors warrants further study to overcome drug resistance in MM cells, including their tumorigenic cancer stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a heterogeneous clonal plasma cell proliferative disorder with CRAB features. Although survival of MM patients has been greatly prolonged by recent implementation of various combinatory treatments with novel anti-MM agents, MM still remains incurable. MM cells preferentially grow and expand in the bone marrow to elicit the alteration of gene expression and thereby drug resistance. To improve the therapeutic efficacy, we urgently need to develop novel treatment strategies targeting the BM microenvironment-mediated drug resistance. The serine/threonine kinase Pim-2 is constitutively over-expressed and acts as a pro-survival mediator in MM cells. We have reported that cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) further up-regulate Pim-2 expression in MM cells to confer drug resistance (Leukemia 2011, 2015). Therefore, Pim-2 appears to be an important therapeutic target to impair the BM microenvironment-mediated drug resistance in MM. Histone deacetylases (HDACs) are generally accepted to be therapeutic targets for MM treatment. However, clinical application of currently available pan-HDAC inhibitors is limited with their adverse effects induced by a non-selective HDAC inhibition. To develop safe and effective HDAC inhibitor-based treatment, the therapeutic roles of HDAC isoform-specific inhibition should be elucidated. In this regard, we have recently reported therapeutic impacts on MM cells of inhibition of class-I HDACs, especially HDAC1 and HDAC3. HDAC3-selective inhibitor BG45 induces anti-MM activity in combination with DNA methyltransferase (DNMT) inhibitor azacytidine (Leukemia 2017). In the present study, we aimed to clarify the underlying mechanisms for impairment of MM cell growth and survival by HDAC1 inhibition. We first referenced the expression of class-I HDACs using a publicly available GSE6691 data set. Among class-I HDACs, HDAC1 and HDAC3 were highly expressed in MM cells. We then knockdowned HDAC1 gene using lentiviral shRNA system in MM cell lines. The HDAC1 gene silencing induced MM cell death with caspase-3 activation, indicating the critical role of HDAC1 in MM cell growth and survival. To determine target molecules of HDAC1, we carried out RNA-sequencing with and without the HDAC1 gene silencing in RPMI 8226 cells. Among genes whose expression significantly changed by the HDAC1 knockdown (adjusted P values 〈 0.05, log fold change 〉 0.5), we focused on IRF4 together with PIM2, because MM cell has been demonstrated to addict to aberrant IRF4-c-Myc regulatory network (Nature 2008). Downregulation of IRF4 and Pim-2 by the HDAC1 knockdown was further confirmed by quantitative PCR (Q-PCR) and immunoblotting in RPMI 8226 and MM.1S cells. Treatment with the class I HDAC-selective inhibitor MS-275 (entinostat) also induced MM cell death along with reduction of IRF4 and Pim-2 expression. Since previous study has shown that IRF4 binds to PIM2 promoter in MM cells (Nature 2008), we examined whether IRF4 regulates PIM2 expression. We found that IRF4 binds to the PIM2 promoter region by analyzing ChIP-Seq data in KMS-12 cells (GSE22901). We further confirmed the binding of IRF4 on PIM2 promoter by ChIP-Q-PCR. Indeed, the IRF4 knockdown downregulated Pim-2 expression in RPMI 8226 cells. These results suggest that HDAC1 inhibition downregulates IRF4 expression, thereby transcriptionally reducing PIM2 expression in MM cells. Pim-2 expression can also be augmented by multiple signaling pathways, including HIF-1a, JAK-STAT and NF-kB-mediated ones in MM cells through the interaction with BM microenvironment. Interestingly, the Pim inhibitor SMI-16a and MS-275 cooperatively induced apoptotic cell death in MM cell lines and CD138-positive primary MM cells even in the presence of BMSCs. Taken together, our results demonstrate the critical role of the HDAC1-IRF4-Pim-2 axis in MM cell growth and survival, and provoke the novel treatment strategy targeting the HDAC1-IRF4-Pim-2 axis in MM cells. Disclosures Anderson: Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.

Description: Survival outcome of patients with multiple myeloma (MM) has been markedly improved by the introduction of novel agents such as bortezomib, thalidomide, and lenalidomide over the past decade. Recent clinical studies have shown further improvement by second-generation agents, namely carfilzomib, ixazomib, pomalidomide, elotuzumab, and daratumumab. However, the prognosis of patients who became refractory to both bortezomib and lenalidomide was extremely poor, and the efficacy of the second-generation agents in such patient remains unclear in routine clinical practice. In this study, we analyzed the outcome of MM patients in terms of overall survival (OS) and survival time after salvage therapy. A total of 174 patients (89 male and 85 female) treated between 2003 and 2017 were enrolled. The median age was 69 years old (range 35-89). The type of M protein was IgG in 93, IgA in 35, IgD in 10, Bence Jones type in 30, and non-secretory type in 5 patients, respectively. The international staging system (ISS) stages I, II, and III were 32, 66, and 63 patients, respectively. There were 100 patients with normal serum LDH and 39 with high LDH. As for initial therapy, 62 patients were treated with conventional chemotherapy, 83 with bortezomib-based regimens, 7 with lenalidomide-based regimens, and 22 with bortezomib + lenalidomide + dexamethasone. Forty-seven patients received autologous stem cell transplantation in the upfront setting. During the observation period 120 patients relapsed and 6 were refractory to initial therapy, and 38 patients were treated with conventional chemotherapy and 58 with the first-generation novel agents such as bortezomib, lenalidomide, or thalidomide as salvage therapy. The remaining 30 patients were further treated with the second-generation novel agents such as carfilzomib in 21, ixazomib in 5, pomalidomide in 6, elotuzumab in 4, or daratumumab in 8 patients after refractory to bortezomib and lenalidomide. The median progression-free survival was 17.7 months and the median OS was 51.0 months from initial therapy. Regarding the outcome according to salvage therapy, the median OS was 22.0 months for conventional chemotherapy group, 46.4 months for first-generation agent group, and 98.7 months for second-generation agent group (p

Description: Multiple myeloma (MM) cells preferentially grow and expand in the bone marrow (BM) to elicit the alteration of gene expression thereby acquiring drug resistance. The serine/threonine kinase PIM2 is constitutively overexpressed which is further up-regulated as a critical anti-apoptotic mediator in MM cells by interacting with BM stromal cells (BMSCs) and/or osteoclasts (Leukemia 2011, 2015). Histone deacetylases (HDACs) generally repress gene expression through deacetylation of lysine residues in histone tails. Therefore, HDAC inhibitors are able to restore the expression of tumor suppressor genes, and utilized as anti-cancer agents for various types of malignancies, including MM. Importantly, class-I and a class-IIb (HDAC6) HDACs have been shown as important therapeutic targets in MM (Nat Chem Biol 2010). Among class-I HDAC isoforms, HDAC1 and HDAC3 are highly expressed in MM cells (GSE5900 and GSE2113) and we have already reported that the HDAC3-DNMT1 axis is a critical therapeutic target (Leukemia 2017). However, the significance of HDAC1 expression in MM cell growth and survival is still largely unknown. In the present study, we aimed to clarify the epigenetic regulation of PIM2 and the therapeutic implication of HDAC1 in MM cells. We observed that HDAC1- and HDAC3-selective inhibitor MS-275 (Entinostat) inhibited MM cell growth in a dose-dependent fashion. HDAC1 knockdown using a lentiviral shRNA system induced apoptosis in MM cell lines, indicating a crucial role of HDAC1 in MM cell growth and survival. To identify downstream targets of HDAC1 mediating MM cell survival, we next carried out RNA-Seq using RPMI 8226 cells after HDAC1 knockdown. Expression of a number of genes were altered (adjusted P values 〈 0.05, log fold change 〉 0.5). Among these genes, we found that PIM2 and IRF4 were significantly downregulated in HDAC1 knocked down cells. The downregulation of IRF4 and PIM2 was further confirmed at mRNA and protein levels in additional MM cell lines. It has been shown that MS-275 impaired the viability of primary MM cells associated with downregulation of IRF4 and PIM2 expression. However, importantly, HDAC1 knocked down-induced growth inhibition was not observed in RPMI8226 cells with IRF4 overexpression, indicating that IRF4 is a key MM cell survival mediator targeted by HDAC1 inhibition. Previous study shows that HDAC1 is abundantly enriched around at H3K27 acetylation or RNA Pol II- binding sites compared to HDAC2 or HDAC3 (GSE86450), However, our data assessed by ChIP-Seq indicated that HDAC1-occupied genes were not completely upregulated but rather downregulated in HDAC1-knockdown cells. Indeed, MS-275 and a histone acetyltransferase inhibitor C646 downregulated IRF4 and PIM2 expression in MM cells despite upregulation and downregulation of histone H3 acetylation, respectively. The ChIP-Seq data showed HDAC1 binding is enriched around the promotor regions of IRF4 and PIM2 in MM cells; however, MS-275 significantly reduced the HDAC1 enrichment as determined in ChIP-Q-PCR assays, suggesting that IRF4 and PIM2 expression is regulated by the balance between acetylation and deacetylation status of histones in MM cells. In addition, we found that IRF4 binds to the promoter of PIM2 and IRF4 knockdown reduced PIM2 expression, suggesting that IRF4 transcriptionally regulates PIM2. Although PIM2 expression is robustly upregulated in MM cells in an ambient microenvironment with BMSCs and/or osteoclasts, MS-275 and the PIM inhibitor SMI-16a cooperatively induce MM cell death. In conclusion, our data provides a basis of rationale combination strategy targeting of class-I HDAC and PIM2 to improve MM patient outcome. Disclosures Anderson: Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board.