ALBERT

Description: Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients' survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. Disclosures No relevant conflicts of interest to declare.

Description: Backgrounds: POEMS syndrome is a rare plasma cell dyscrasia presenting with polyneuropathy, l-type M protein, VEGF elevation and systemic manifestations. The standard treatment of the disease has not been established through clinical trials, but autologous stem cell transplantation (ASCT) has been shown to be effective in patients with the syndrome. However, the efficacy and long-term outcomes of POEMS patients underwent ASCT have not been systematically studied. Purpose and Methods: To clarify the efficacy and long-term outcomes of the patients underwent ASCT, we performed a multicenter retrospective study assessing the clinical course of patients registered to the Japan Society for Hematopoietic Cell Transplantation (JSHCT) TRUMP database. The definition and evaluation of response and progression/relapse was according to the criteria of Mayo Clinic (Blood.2012; 120:56-62). Results: Between January 2000 and December 2011, 94 patients were registered to the TRUMP database, male 57 and female 37, with the median age of 52 years (range 28-72). All patients underwent PBSCT except for one who received both PBSCT and BMT. Two patients underwent 2nd ASCT. The median time to ASCT from the time of diagnosis of POEMS was 205 days (range: 16-3870 days). Peripheral blood stem cells were collected using either cyclophosphamide with G-CSF, G-CSF alone, or etoposide with G-CSF as mobilizing agents with a median of 1 apheresis procedure. Thirteen of the 68 patients (19.1%) needed additional harvest procedures, and two patients received G-CSF with plerixafor. Median total collected CD34+ cell dose was 4.0x106/kg (0.6-37.2x106/kg).The conditioning regimen was melphalan in 93/94 (99%): 68 patients (70.8%) received melphalan ≥ 200 mg/m2. Median infused CD34+ cell dose was 3x106/kg (0.02-20x106/kg). Periengraftment syndrome was recognized in 11 of the evaluable 68 patients (16.2%). After ASCT, patients' performance status (PS) was dramatically improved (ECOG PS 3-4; 33.7% to 8.2%). The best initial responses were as follows: hematologic response; CRH/VGPR H/PRH 38/51 (74.5%), NR H 13/51 (25.4%), clinical response; CR/PR 56/61 (91.2%), SD 4/61 (6.6%), PD 1/61 (1.6%), VEGF response; CRv/PRv 34/35 (97.1%), NRv 1/35 (2.9%). 17 patients relapsed/progressed after ASCT. Four patients had radiographic relapse, 7 patients had symptomatic progression, and 10/12 (83.3%) patients had elevated VEGF. There was no hematologic relapse/progression. With the median follow up of 26 months (range 1-102 months), 10 patients died and the overall survival at 4 years was 89.9%. Sixteen patients received post-transplant treatment (planned tandem ASCT:1, steroids: 7, Bor: 1, Thal: 11, Ren: 9, VAD: 1, RT: 1). The causes of death were; disease progression (4), infection (3; PCP 1, bacterial 2), and others (3). Conclusion: We show the long-term outcomes of patients with POEMS syndrome treated with ASCT. These data suggest the promising role of ASCT in patients with POEMS syndrome both in producing prolonged survival and improving quality of life. Figure 1. Figure 1. Kaplan-Meier analysis for OS post SCT Disclosures No relevant conflicts of interest to declare.

Description: The expression of NR4A3, which is a member of the gene encoding NR4A orphan nuclear receptor subfamily, has been reported to be commonly silenced in blasts of patients with acute myeloid leukemia (AML), irrespective of karyotype. In line with this finding, Nr4a1-/-/Nr4a3-/- mice rapidly develop AML within one month following birth (Mullican et al., 2007). In addition, Nr4a1+/-/Nr4a3-/- and Nr4a1-/-/Nr4a3+/- mice show myelodysplastic/myeloproliferative neoplasms (Ramirez-Herrick et al., 2011), suggesting that NR4A3 functions as a tumor suppressor gene in myeloid malignancies. The extremely short latency of AML development in Nr4a1-/-/Nr4a3+/- mice indicates that silencing these tumor suppressors is sufficient to induce AML and that NR4A3 has a crucial role in the pathogenesis of AML. Thus, unveiling the molecular mechanism that regulates NR4A3 expression in AML would facilitate the development of novel therapies, including transcriptional reactivation of the gene. However, the therapeutic modalities targeting NR4A3 have been hindered by our minimal understanding of the mechanism underlying reduced NR4A3 expression, particularly in human AML cells. Abnormal epigenetic regulation is a common mechanism in the pathogenesis of several types of cancers. For instance, the expression of several tumor suppressor genes, such as p16 and MLH1, is repressed due to DNA hypermethylation at their promoter regions. Given that loss-of-function mutations in NR4A3 have not been reported in AML to date, we hypothesized that DNA hypermethylation contributes to a reduction in NR4A3 expression in AML. To test our hypothesis, we analyzed DNA methylation status of NR4A3 in human AML cells. We first compared the level of NR4A3 expression in eight human AML cell lines and two human primary AML samples, with that in CD34+ mononuclear bone marrow (BM) cells from healthy human controls. As expected, the expression of NR4A3 was markedly reduced in all of the AML cell lines and primary AML cells compared with that in the cells of the healthy controls. To evaluate the function of NR4A3 in human AML cells, we ectopically overexpressed NR4A3 in a human AML cell line (NB4 cells). The growth of NR4A3 -overexpressing NB4 cells was remarkably compromised compared with that of the controls, suggesting a tumor suppressive function of NR4A3 in both human AML and murine cells. To investigate the DNA methylation status of NR4A3, we performed bisulfite sequencing assays using eight human AML cell lines (HL60, NB4, Kasumi, TF1, U937, K562, MOLM13, and THP1) as well as CD34+ BM cells from healthy controls. Unexpectedly, a hypermethylated CpG site in the promoter region was not detected in any of the cell lines. However, the drastically or mildly methylated region including twenty eight CpGs was identified approximately 3 kb downstream of the transcription start site in six AML cell lines (97.5%, 78.3%, 77.1%, 89.9%, 95.2%, and 86.9% in HL60, NB4, Kasumi, TF1, U937, and K562, respectively) and two mixed lineage leukemia-related cell lines (31.0% and 53.6% in MOLM13 and THP1, respectively), whereas this site was hypomethylated in the controls (n = 2; mean, 12.7%; range, 7.1%-18.2%). To evaluate the contribution of this hypermethylated region to reduced NR4A3 expression, the six AML cell lines with heavily hypermethylated CpGs at NR4A3 and two human primary AML cell samples were treated with a DNA methyltransferase inhibitor (decitabine; DAC) for three or five days. DAC exposure inhibited cell growth and restored the expression of NR4A3 in all AML cell lines and primary cells in a dose- and time-dependent manner. Next, we examined the status of DNA methylation at the CpG site following DAC treatment with bisulfite sequencing assays. The frequencies of methylated CpG in HL60, NB4, and K562 cells was reduced from 97.5% to 53.6%, 78.3% to 68.5%, and 86.9% to 67.5% after DAC treatment, respectively. In contrast, the methylation status in Kasumi, TF1, and U937 cells did not significantly changed after DAC treatment. Our findings in the present study suggest that DNA hypermethylation may partially account for the transcriptional inactivation of NR4A3 in AML. However, the mechanism of reduced NR4A3 expression is complex and variable depending on the genetic background. We are currently working on a more detailed analysis of DNA methylation using human primary cells, by extending the regions for investigation, such as enhancer regions. Disclosures Nakaseko: Novartis: Honoraria, Research Funding, Speakers Bureau; Otsuka: Honoraria, Research Funding; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.