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SUMMARYPolycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and β-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

Among the most important histone methyltransferases for metazoan development are EZH1/2 and their homologs, which methylate histone H3 lysine 27 and act as part of a highly conserved set of chromatin regulators called Polycomb Group (PcG) proteins. Reaching a precise understanding of the roles of PcG proteins in the orchestration of differentiation and the maintenance of cell identity requires a variety of genetic and molecular approaches. Here, we present a full suite of methods for the study of PcG proteins in early murine development, including mutant strain generation, embryonic stem cell derivation, epigenomic profiling, and immunofluorescence and in situ hybridization.

Renal manifestations of multiple myeloma (MM) including cast nephropathy, amyloidosis, and renal calcification have been widely recognized. However, the severity of histopathological findings has not been addressed so far, and the clinical significance of these pathological findings is unclear. We sought to clarify the relationship between the severity of renal pathology and clinical characteristics. We analyzed 53 autopsies performed on patients who died from MM. The kidneys were evaluated using light microscopy, and the severity of pathological findings was recorded. The most common renal lesion was cast nephropathy (n = 27). Other findings included amyloidosis (n = 10), renal calcification (n = 5), microbial infection (n = 4), and MM infiltration (n = 17). The incidence of MM infiltration was substantially higher than previously reported. Renal MM infiltration was detected even when bone marrow plasmacytosis was limited. However, a significantly higher degree of renal MM infiltration was observed when MM cells invaded the liver. No correlation was observed between serum creatinine levels and degree of MM infiltration, but these tended to be elevated when cast nephropathy was severe. These findings may provide clues to understand both renal injury and extramedullary diseases in patients with MM.

Extramedullary disease (EMD) is an issue for patients with multiple myeloma (MM), since extramedullary spread of MM is associated with an aggressive course and a poor prognosis. Moreover, the mechanism of EMD development is uncertain. Here, we present extensive extramedullary plasmacytoma occupying the left upper limb of a 66-year-old female patient with MM with an extremely aggressive course and multiple visceral organ involvement without bone marrow infiltration or plasma cell leukemia. EMD of this large size is extremely rare and this case may provide a clue for better understanding of clinical features of EMD in MM.

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the “time-lapse” TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%–30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.

Polycomb repressive complex (PRC) resides in two major complexes PRC1 and PRC2. They cooperate with each other to coordinate proper developmental process by silencing target genes; PRC1 posits H2AK119ub1 and PRC2 catalyzes trimethylation of H3K27 (H3K27me3). The PRC1 component BMI1/PCGF4 has long been recognized to be essential in the maintenance of normal and malignant hematopoietic stem cells (HSCs). Recently, diversity of PRC1 has been noticed and PRC1 is now classified into six alternative complexes depending on PCGF proteins. In embolic stem cells, PRC1 which contains PCGF1 (PCGF1-PRC1) has been demonstrated to serve upstream of the BMI1/PCGF4-PRC1. However, the impact of BCOR, which is a component of PCGF1-PRC1 on hematopoiesis is clearly different from BMI1/PCGF4; the mice deficient for BCOR exhibited normal HSC activities and BCOR rather prevented leukemic transformation of HSCs, suggesting the previously unappreciated gene control mechanisms of PCGF1-PRC1. To tackle this issue, we focused on the roles of PCGF1 in hematopoiesis. Loss of Pcgf1 in hematopoietic stem cells led to severe reduction of B lineage cells with an expansion of myeloid progenitors due to defects in lymphoid-primed multipotent progenitor (LMPP) cells. To explore the molecular mechanisms, we have established Id3-overexpressing hematopoietic progenitor cells (IdHPs) which correspond to LMPP-like cells (Ikawa et al. 2015) from bone marrow of ERT2-Cre Pcgf1 flox mice. The ChIP-seq analysis of normal IdHPs identified 1274 genes whose promoters were associated with PCGF1 peaks and 37% of them exhibited enrichment of H3K27me3 and binding of SUZ12 (PRC2). Deletion of Pcgf1 destabilized H3K27me3 levels, resulting in re-activation of genes associated with PCGF1 and SUZ12 peaks, whereas the chromatin occupancy of SUZ12 was not affected. Intriguingly, proteomic analysis demonstrated that PCGF1 interacts with key factors responsible for the organization of nucleosomes and PCGF1 loss triggered a decline of nucleosome-densities in promoters of genes occupied by PCGF1 and SUZ12 peaks. Since enzymatic activity of PRC2 is dependent on nucleosome-densities, PCGF1 is likely to regulate the susceptibility of H3K27me3 by PRC2 through determination of the nucleosome-densities. Furthermore, genes which were downregulated by PCGF1-nucleosome-H3K27me3 axis entailed many myeloid-related genes and knock down of one of those myeloid genes partially restored the B cell differentiation potential of Pcgf1-KO hematopoietic stem/progenitor cells (HSPCs), supporting the biological significance of the PCGF1-nucleosome-H3K27me3 axis. Collectively, these results indicate PCGF1 determines cellular fate of HSPCs through stabilization of nucleosomal organization. Disclosures No relevant conflicts of interest to declare.

Second primary malignancies (SPMs) are issues for patients with multiple myeloma (MM). There may have been some limitations in prior studies, such as difficulties in a longer follow-up and absence of established screening methods. Therefore, we studied autopsied cases to overcome these limitations. This study aimed to examine SPMs using autopsy reports. Ninety-one cases of MM autopsied at our institution from 1979 to 2013 were analyzed. Median age of autopsied patients was 64.1 years, and proportion of male/female was 59/32. Autopsy was performed in 35.3% of patients died of MM. There were five cases of SPMs with a median confirmation time of 38 (12-132) months from the diagnosis of MM. In three of the five patients, the diagnosis of SPMs was established at autopsy. One case was of myelodysplastic syndrome, and the others were of non-hematological malignancies. The annual risk of SPM estimated using the Kaplan-Meier method was approximately 1%. Three of five SPM cases were detected at autopsy. Analysis of autopsy may contribute to estimate the actual risk of SPMs in MM.

12 p.-6 fig.1 tab.Tomokatsu Ikawa, et al., In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5. These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program., This work was supported in part by grants from the Japan Society for the Promotion of Science (24689042 to T.I.), the Japan Science and Technology Agency (T.I.), RIKEN Center for Integrative Medical Sciences (IMS) Young Chief Investigator program (T.I.), and the Kanae Foundation for the Promotion of Medical Science (T.I.).

The safety and efficacy of treatment with liposomal amphotericin B (L-AMB) in elderly patients has not been clarified, especially in Japanese patients. Therefore, we retrospectively analyzed 33 elderly patients with hematological diseases of at least 65 years old who received L-AMB between 2009 and 2012. Their clinical outcomes were compared to those of 21 patients who were younger than 65 years. L-AMB was administered for empirical therapy (n = 2) or target therapy for possible (n = 14) or probable/proven (n = 17) invasive fungal infection. There was no discontinuation of L-AMB due to adverse events. More than 2-fold increases from the baseline Cre, AST, and ALT values were observed in 21.2%, 39.4%, and 45.5% of the older group and 38.1%, 61.9%, and 52.4% of the younger group, respectively. The concurrent use of nephrotoxic antibiotics was the only risk factor for the development of a 2-fold increase in the serum Cre level. The duration of L-AMB was significantly longer in patients who developed grade III-IV hypokalemia. A partial or complete response was observed in 54.8% and 62.5% of the elderly and younger groups, respectively. In conclusion, L-AMB therapy appeared to be acceptably safe as empirical therapy or treatment for invasive fungal infection.

Background Renal injury is a common complication in multiple myeloma (MM). Myeloma cast nephropathy is the most common lesion and this is caused by excessive light chains binding to uromodulin. Other than cast nephropathy, renal calcification, renal amyloidosis, and direct tumor cell involvement have been noticed. However seldom had previous reports addressed the issue of severity of histopathologic findings. Therefore, this study investigates the extent of each renal pathological finding, and examines the correlation of such severity and clinical findings in autopsied cases. This may offer understanding of renal injury in patients with MM from another angle, since similar approach had rarely been taken. Patients and methods Fifty-three sequential autopsies performed on patients died of MM at our hospital, were analyzed. The kidneys were evaluated by light microscopy using hematoxilin-eosin-stained sections as well as Congo red stain when amyloidosis was suspected. When the cast nephropathy and/or invasion of MM cells are identified, we described their severity. In the case of cast nephropathy, if the cast is formed in the fraction of the tubules they are defined as class1, if the casts are existed diffusely, they are class2, and if the diffuse casts are associated with interstitial atrophy, they are class3. When local MM invasion was found, this was defined as class1, diffuse involvement class2, and when the basic constitution of nephron is collapsed by MM involvement they are class3. Other histopathologic findings such as renal calcification, microbial and fungal infection, micro thrombo-embolism, and arteriosclerosis were also recorded. Results Patients, characteristics are shown in the table. Median age at autopsy was 64.5 years old. Durie & Salmon stage: Almost all cases were at stage III, M-protein: IgG/IgA/IgD/BJP/uncertain=26/10/3/11/3 cases. Median overall survival was 49 months. Seven of 53 patients manifested as plasma-cell leukemia. The most common renal lesion was cast nephropathy (49.9%). We found 63.4% of them were categorized as class 1, 28.4% class 2, 9.2% class 3. Plasma cell tumor involvement was found in 30.0% of 53 cases. Seventy nine point six per cent of them were class 1, 2.4% class 2, 18.0% class 3. Other findings include, Al-amyloidosis (17.1%), renal calcification (10.1%), microbiological and fungal infection (7.7%), micro thrombo-embolism (7.7%). Thirty-three per cent of the patients had the glomerular sclerosis which involve more than 20“ of the glomerulus (Figure. 1). As shown in the Figure. 2 the severity of cast-nephropathy was significantly correlated with serum creatinine level (r=0.278, p=0.048). Longer overall survival was found in patients who hadrenal involvement of the kidney (56.5 months vs 46.8 months. p=0.012). MM cells invaded more profoundly when there was evidence of plasma-cell leukemia (class 1.43 vs class 0.41. p Discussion This study clearly showed that cast nephropathy was the most common renal manifestation. This was comparable with previous reports. However, there were larger numbers of cases with myeloma invasion in kidney than expected, and more profound MM invasion was found in cases with plasma-cell leukemia. This indicated that MM can directly invade kidney in the end-stage of MM. Long term survivors tended to have increased incidence of renal direct invasion of MM. This may reflect the natural history of MM. We found correlation between severity of cast nephropathy and serum creatinine. Together, these findings may provide the framework for future direction. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Abstract 2007 Background: High-dose cyclophosphamide (HD-CY) + granulocyte-colony stimulating factor (G-CSF) and G-CSF alone have been used to mobilize hematopoietic stem cells (HSCs) for autologous SC transplantation (ASCT) in multiple myeloma (MM). However, which regimen is better is unknown; anti-myeloma effects of HD-CY + G-CSF have not been established. From January 1999 to June 2009, we administered HD-CY+G-CSF but changed to G-CSF alone during July 2009–December 2010. We retrospectively assessed HSC collection efficacy, complications, and anti-myeloma effects of these regimens. Patients and methods: We analyzed 147 MM patients from whom HSCs were to be collected at our institute. For mobilization, 115 patients were administered HD-CY (4 g/m2)+G-CSF (600 mg/body filgrastim or 500 mg/body lenograstim) and 32 were administered G-CSF alone (same dose as HD-CY). Here, 17 patients received therapeutic intervention between mobilization and transplantation without disease progression (PD). To avoid the patient outcome effect, we defined event- and progression-free survivals (EFS and PFS). EFS was defined as PD, death, or therapeutic intervention without PD. PFS was defined as PD or death, where therapeutic intervention without PD was used as a censor. Both were calculated from the start of mobilization. For analyzing response by mobilization, patients receiving therapeutic intervention without PD were excluded. Response was evaluated in those not receiving therapeutic intervention without PD or in whom response could not be evaluated before ASCT. Thalidomide was administered as maintenance therapy to 14 and 6 patients in the HD-CY+G-CSF and G-CSF groups after ASCT. Thalidomide administration was used as a censor. Results: Vincristine, doxorubicin, and dexamethasone (VAD) and HD dexamethasone (HDD) therapies were administered as induction therapy (VAD for 117, HDD for 2, and both for 11). New (bortezomib or thalidomide) and alkylating agents were administered to 7 and 13 patients, respectively. Before mobilization, 26 patients received radiotherapy; none were administered lenalidomide. No statistical difference was seen in baseline characteristics (Durie-Salmon stage, International staging system, interval from diagnosis to mobilization, disease control, and previous therapies) between both groups. However, patients mobilized by G-CSF alone were significantly older. Among 147 patients, 121 underwent planned ASCT. Of the 17 receiving therapeutic intervention without PD, 13 and 4 belonged to the HD-CY+G-CSF and G-CSF groups, respectively. More than 2 × 106 CD34-positive cells/kg were collected from 93% and 75% patients in the HD-CY+G-CSF and G-CSF (p = 0.0079) groups, respectively. More than 4 × 106 CD34-positive cells/kg were collected from 84% and 69% in the HD-CY+G-CSF and G-CSF (p = 0.07). Mean HSC count was 11.4 × 106/kg in the HD-CY+G-CSF group and 4.5 × 106/kg in the G-CSF group (p = 0.0007). Among patients receiving HD-CY+G-CSF, 66% were treated with intravenous antibiotics; 3 suffered cardiac shock and 2 septic shock. However, among those receiving G-CSF alone, no severe complications were seen. Median hospitalization days were 21 and 8 for the HD-CY+G-CSF and G-CSF groups, respectively (p < 0.0001). In the HD-CY+G-CSF group, 16% improved in disease control before ASCT, 71% showed no change, and 13% progressed. However, no patient improved, 63% showed no change, and 27% progressed in the G-CSF group (p = 0.015). Median EFS was 25 months in the HD-CY+G-CSF group and 13 in the G-CSF group (fig 1, p value of log-rank test = 0.012). Median PFS was 28 months in the HD-CY+G-CSF group and 15 in the G-CSF group (fig 2, p value of log-rank test = 0.011). Median overall survival did not differ significantly. Conclusion: Regarding the safety and duration of hospitalization, G-CSF alone may be safer and beneficial. However, HD-CY+G-CSF was more effective as a mobilization regimen and showed higher anti-myeloma effects than G-CSF alone. Disclosures: No relevant conflicts of interest to declare.

Abstract 5001 Background: Renal insufficiency is one of the main complications in myeloma patients. Various causes were reported to be responsible for renal damage. Through analysis of an autopsied cases we would show the diversity of the renal diseases in myeloma. Methods: We studied 41 autopsied myeloma cases from 1979 to 2008 at the National Center for Global Health and Medicine. The kidneys were evaluated by light microscopy using hematoxilin-eosin-stained sections, as well as Congo-red stain when amyloidosis was suspected. Results: There were 21 men and 20 women. Mean age at autopsy was 64.5 years old. The most common lesion was cast nephropathy (41.5%). The giant cell invasion was found in 35.3% of patients with cast nephropathy. Plasma cell tumor involvement was detected in 29.3% of all 41 autopsied cases. Fourteen per cent of cases had both cast nephropathy and plasma cell tumor involvement. In 75.6% of the patients, arteriosclerosis was found. In addition, 32% of the patients had the glomerular sclerosis which involved more than 20% of the glomerulus. Other findings include acute tubular necrosis (31.7%), AL-amyloidosis (19.5%), renal calcification (17.1%), bacterial and fungal infection (7.7%), micro thrombo-embolism (7.7%), mesangial proliferation (4.9%). At least one of above findings were detected in all cases and combined findings were detected in 65.9% cases. Conclusion: We evaluated the renal manifestations in 41 autopsied cases in detail. In all cases, at least one pathological finding was detected. Cast nephropathy was the most common renal manifestation. However, direct myeloma cell invasion was found in 29.3% and combined pathologic findings were common. Disclosures: No relevant conflicts of interest to declare.