Search

Multiple myeloma remains incurable due to the persistence of a minor population of multiple myeloma cells that exhibit drug resistance, which leads to relapsed and/or refractory multiple myeloma. Elucidating the mechanism underlying drug resistance and developing an effective treatment are critical for clinical management of multiple myeloma. Here we showed that promoting expression of the gene for polycomb-like protein 3 (PHF19) induced multiple myeloma cell growth and multidrug resistance in vitro and in vivo. PHF19 was overexpressed in high-risk and drug-resistant primary cells from patients. High levels of PHF19 were correlated with inferior survival of patients with multiple myeloma, in the Total Therapy 2 cohort and in the Intergroup Francophone du Myeloma (IFM) cohort. Enhancing PHF19 expression levels increased Bcl-xL, Mcl-1, and HIF-1a expression in multiple myeloma cells. PHF19 also bound directly with EZH2 and promoted the phosphorylation of EZH2 through PDK1/AKT signaling. miR-15a is a small noncoding RNA that targeted the 3′UTR of PHF19. We found that downregulation of miR-15a led to high levels of PHF19 in multiple myeloma cells. These findings revealed that PHF19 served a crucial role in multiple myeloma proliferation and drug resistance and suggested that the miR-15a/PHF19/EZH2 pathway made a pivotal contribution to multiple myeloma pathogenesis, offering a promising approach to multiple myeloma treatment.Implications:Our findings identify that PHF19 mediates EZH2 phosphorylation as a mechanism of myeloma cell drug resistance, providing a rationale to explore therapeutic potential of targeting PHF19 in relapsed or refractory patients with multiple myeloma.

Supplementary Fig.1. PHF19 gene expression is associated with poor prognosis in myeloma patients. Supplementary Fig.2. Enhanced PHF19 expression in OCI-My5 cells decrease drug sensitivity. Supplementary Fig.3. PHF19 interacts with EZH2. Supplementary Fig.4. PHF19 promotes EZH2 phosphorylation. Supplementary Fig.5. RNA sequencing indicates that PHF19 activates proliferation-related signaling pathways. Supplementary Fig.6. PHF19 promotes EZH2 phosphorylation via AKT pathway. Supplementary Fig.7. miR-15a can downregulation PHF19 expression. Supplementary Fig.8. Visual Overview

Purpose:That the malignant clone of Waldenström's macroglobulinemia (WM) demonstrates significant intraclonal heterogeneity with respect to plasmacytoid differentiation indicates the mechanistic complexity of tumorigenesis and progression. Identification of WM genes by comparing different stages of B cells may provide novel druggable targets.Experimental Design:The gene expression signatures of CD19+ B cells (BC) and CD138+ plasma cells (PC) from 19 patients with WM were compared with those of BCs from peripheral blood and tonsil and to those of PCs from the marrow of healthy (N-PC) and multiple myeloma donors (MM-PC), as well as tonsil (T-PC). Flow cytometry and immunofluorescence were used to examine T-cell marker expression on WM tumor cells.Results:Consistent with defective differentiation, both BCs and PCs from WM cases expressed abnormal differentiation markers. Sets of 55 and 46 genes were differentially expressed in WM-BC and WM-PC, respectively; and 40 genes uniquely dysregulated in WM samples were identified. Dysregulated genes included cytokines, growth factor receptors, and oncogenes not previously implicated in WM or other plasma cell dyscrasias. Interestingly, strong upregulation of both IL6 and IL6R was confirmed. Supervised cluster analysis of PC revealed that marrow-derived WM-PC was either MM-PC–like or T-PC–like, but not N-PC–like. The aberrant expression of T-cell markers was confirmed at the protein level in WM-BC.Conclusions:We showed that comparative microarray profiles allowed gaining more comprehensive insights into the biology of WM. The data presented here have implications for the development of novel therapies, such as targeting aberrant T-cell markers in WM.

Supplemental Data: four Tables and one Figure Supplement Table 1. B-cell markers and differentiation-related genes in WM, MM, and their normal counterparts Supplement Table 2. Most differentially expressed genes (n = 55) allowing the distinction of WM-BC from PB-BC and T-BC Supplement Table 3. Most differentially expressed genes (n = 46) allowing the distinction of WM-PC from T-PC and N-PC Supplemental Table 4. Clinical information of WM patients used for the Figure 4 Supplemental Fig. 1: Immunofluorescence shows CD3 expression (Green) on normal T cells and CD19 expression (Red) on normal B cells derived from peripheral blood of a healthy donor. DAPI (blue color) was used to stain cell nuclei.

Key Points The clonal architecture of MM at relapse was identified using QM-FISH at the single-cell level.The prognostic values of different clonal evolution patterns were explored in large-scale clinical analysis., Visual Abstract, Single-cell analysis is of significant importance in delineating the exact phylogeny of the subclonal population and in discovering subtle diversification. So far, studies of intratumor heterogeneity and clonal evolution in multiple myeloma (MM) were largely focused on the bulk tumor population level. We performed quantitative multigene fluorescence in situ hybridization (QM-FISH) in 129 longitudinal samples of 57 MM patients. All the patients had newly diagnosed and relapsed paired samples. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. Forty-three of 57 patients (75.4%) harbored 3 or 4 cytogenetic clones at diagnosis. We delineated the phylogeny of the subclonal tumor population and derived the evolutionary architecture in each patient. Patients with clonal stabilization had a significantly improved overall survival (OS) than those with other evolutionary patterns (median OS, 71.2 months vs 39.7 months vs 35.2 months vs 25.5 months, for stable, differential, branching, and linear patterns, respectively; P = .001). A high degree of consistency and complementarity across QM-FISH and cFISH was observed in the evaluation of cytogenetic evolution patterns in MM. Survival after relapse was greater influenced by the presence of high-risk aberrations at relapse (hazard ratio = 2.07) rather than present at diagnosis (hazard ratio = 1.55). This study shows that QM-FISH is a valuable tool to elucidate the clonal architecture at the single-cell level. Clonal evolution pattern is of prognostic significance, highlighting the need for repeated cytogenetic evaluation in relapsed MM.

Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Proteasome inhibitors (PIs) work as the backbone agent and have greatly improved the outcome in majority of newly diagnosed patients with myeloma. However, drug resistance remains the major obstacle causing treatment failure in clinical practice. Here, we investigated the effects of Indirubin-3'-monoxime (I3MO), one of the derivatives of Indirubin, in the treatment of MM.MM patient primary samples and human cell lines were examined. I3MO effects on myeloma treatment and the underling molecular mechanisms were investigated via in vivo and in vitro study.Our results demonstrated the anti-MM activity of I3MO in both drug- sensitive and -resistance MM cells. I3MO sensitizes MM cells to bortezomib-induced apoptosis. Mechanistically, I3MO acts as a multifaceted regulator of cell death, which induced DNA damage, cell cycle arrest, and abrogates NF-κB activation. I3MO efficiently down-regulated USP7 expression, promoted NEK2 degradation, and suppressed NF-κB signaling in MM. Our study reported that I3MO directly bound with and caused the down-regulation of PA28γ (PSME3), and PA200 (PSME4), the proteasome activators. Knockdown of PSME3 or PSME4 caused the inhibition of proteasome capacity and the overload of paraprotein, which sensitizes MM cells to bortezomib-mediated growth arrest. Clinical data demonstrated that PSME3 and PSME4 are over-expressed in relapsed/refractory MM (RRMM) and associated with inferior outcome.Altogether, our study indicates that I3MO is agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM.A full list of funding can be found in the acknowledgements.

Farmer paintings are an important part of the Chinese cultural system. After decades of development, Farmer Painting has formed its own artistic characteristics and industrial development model. With the development of computer drawing technology, the traditional peasant painting creation mode has been challenged unprecedentedly. In order to better promote the development of Chinese farmer painting industry, this paper studies the development trend of Chinese farmer painting based on computer drawing technology. This article conducts an in-depth study of the two existing creative modes of Chinese farmer paintings. According to the survey results, traditional farmer paintings pay more attention to creativity and artistry. However, after the complete marketization, the quality of the paintings has seriously declined, but the income has increased significantly. In response to this situation, this article analyzes that digitization and industrialization are the main development trends of peasant paintings, but excessive marketization will damage the artistry of peasant paintings and hinder its development. Therefore, in response to these problems and the current situation of computer rendering technology in farmer painting creation and the needs of the development of the digital industry, combined with the current development of the Internet, a set of improvement measures have been proposed. Specifically, from the three aspects of refining the production process, the government's efforts to control development, and the formulation of industry access. The analysis shows that improvement measures can resolve the contradiction between the industrialization and artistry of farmer paintings and establish a long-term management mechanism.

Pulsed dye laser (PDL) is the gold-standard therapy for port-wine stains (PWSs), but the therapeutic efficacies vary widely with each individual. Recent studies suggest that hematoporphyrin monomethyl ether-photodynamic therapy (HMME-PDT) is an effective alternative in the treatment of PWS. This retrospective study aimed to compare the effects of HMME-PDT and Cynergy dual-wavelength laser on red and purple color types of PWS METHODS: This retrospective study included 38 PWS patients who received Cynergy dual-wavelength laser treatment (PDL group) and 39 PWS patients who received HMME-PDT treatment (HMME-PDT group). Patients in each group received two treatments. The curative effect of purple or red PWS in two groups was analyzed.After the first treatment, the total significant response rate and the total response rate of the HMME-PDT group were 43.59% and 74.36%, which were significantly higher than those of the PDL group (5.26% and 47.36%) (P0.05). After the second treatment, the total significant response rate and the total response rate of the HMME-PDT group were 69.23% and 100%, which were obviously higher than those of the PDL group (34.21% and 68.42%) (P0.05). For both groups, the total significant response rate of purple-type PWS was remarkably higher than that of red-type PWS (the HMME-PDT group: P = 0.004; the PDL group: P = 0.048). There was no obvious difference in terms of patient age and PWS location between the HMME-PDT and PDL groups. Furthermore, there was no considerable difference in the incidence of adverse reactions between the two groups (P0.05).The single-center retrospective study suggested that HMME-PDT might have better efficacies on purple and red PWS than PDL treatment. The total treatment efficacy was greater in purple-type PWS than in red-type PWS.

Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Proteasome inhibitors (PIs) work as the backbone agent and have greatly improved the outcome in majority of newly diagnosed patients with myeloma. However, drug resistance remains the major obstacle causing treatment failure in clinical practice. Indirubin-3’-monoxime (Id-3) is one of the derivatives of Indirubin, a traditional Chinese medicine that has reported to be effective in the treatment of chronic myeloid leukemia and some solid tumors. In this study, we demonstrated the anti-MM activity of Id-3 in both drug sensitive and bortezomib-resistance human MM cell lines and patient-derived cells. Moreover, combination with Id-3 could sensitize MM cells to bortezomib-induced apoptosis. Mechanistically, Id-3 acts as a multifaceted regulator of cell death, which induces DNA damage and cell cycle arrest as well as abrogates NF-κB activation in MM cells. Id-3 efficiently down-regulates USP7 expression, resulting in destabilization of NEK2 and suppression of NF-κB signaling. Importantly, proteasome complex subunits PSME3 (PA28γ) and PSME4 (PA200) were down-regulated in MM cells treated with Id-3. Knockdown of PSME3 or PSME4 by shRNA caused proteasome inhibition and paraprotein accumulation, as well as sensitized MM cells to bortezomib-mediated growth arrest. Clinical data analysis demonstrated that PSME3 and PSME4 are over-expressed in relapsed/refractory MM and associated with inferior overall survival. Taken together, our study indicate that Id-3 is a novel agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM. Funding: This work was supported by the Natural Science Foundation of China (8217011925, 81570181, 81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006, 2018RC320012), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences CAMS- 2017-I2M-1-005, 2016-I2M-3-023). National Program on Key Basic Research Project 2018ZX09733003. Declaration of Interest: Dr. Kenneth. C. Anderson is the consultant: Pfizer, Amgen, Astrazeneca, Janssen, Precision Biosciences, Mana and Window. Founder/Stock Shareholder: C4 Therapeutics, Oncopep, Raqia and NextRNA. Other authors declared no competing financial interests in relation to the work described. Ethical Approval: The study was approved by the Medical Ethical Committee of the Institute of Hematology & Blood Diseases Hospital, State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China.

Anaemia is the most common complication of myeloma and is associated with worse clinical outcomes. Although marrow replacement with myeloma cells is widely considered a mechanistic rationale for anaemia, the exact process has not been fully understood. Our large cohort of 1363 myeloma patients had more than 50% of patients with moderate or severe anaemia at the time of diagnosis. Anaemia positively correlated with myeloma cell infiltration in the bone marrow (BM) and worse patient outcomes. The quantity and erythroid differentiation of HSPCs were affected by myeloma cell infiltration in the BM. The master regulators of erythropoiesis, GATA1 and KLF1, were obviously downregulated in myeloma HSPCs. However, the gene encoding the chemokine CCL3 showed significantly upregulated expression. Elevated CCL3 in the BM plasma of myeloma further inhibited the erythropoiesis of HSPCs via activation of CCL3/CCR1/p38 signalling and suppressed GATA1 expression. Treatment with a CCR1 antagonist effectively recovered GATA1 expression and rescued erythropoiesis in HSPCs. Myeloma cell infiltration causes elevated expression of CCL3 in BM, which suppresses the erythropoiesis of HSPCs and results in anaemia by downregulating the level of GATA1 in HSPCs. Thus, our study indicates that targeting CCL3 would be a potential strategy against anaemia and improve the survival of myeloma patients.

The research on antifungal resistance in dermatophytoses lags behind that on systemic mycose. Lack of datas of antifungal susceptibility testing in dermatophytoses is one reasion. 121 clinical dermatophytes isolates were tested against 6 azole antifungal agents according to the Clinical and Laboratory Standards Institute (CLSI) method. Geometric mean MIC of all isolates were in increasing order: isavuconazole (GM 0.06 mg/L), posaconazole (GM 0.10 mg/L), itraconazole (GM 0.22 mg/L), voriconazole (GM 0.32 mg/L), ketoconazole (GM 0.40 mg/L), fluconazole (GM 10.18 mg/L).

Chromosome 1q21 aberrations in multiple myeloma have attracted much attention for a long time, however, the prognostic value is still under investigation. We confirmed the independent prognostic impact of 1q21 aberrations in this non-randomized clinical study. Our study noted that additional copies and larger clonal size of 1q21 gain did not worsen the outcome. We discovered that 1q21 gain was associated with the acquisition of new chromosome abnormalities and genomic instability, evidenced by the strong correlation between 1q21 gain and complex karyotypes or the acquisition of more than two cytogenetic aberrations. Moreover, 1q21 gain and/or del(17p) were powerful enough to discriminate high-risk patients. Furthermore, 1q21 gain retained unfavorable even when stratified by concurrent presence of t(4;14), especially in the bortezomib arm. Finally, although bortezomib might benefit patients with 1q21 gain, it could not completely overcome its adverse effects, suggesting the necessity of more effective therapies for these patients.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Although the use of bortezomib (BTZ) significantly improves MM therapy, intrinsic and acquired drug resistance to BTZ remains a major clinical problem. In this study, we find that Cdc37, a key co-chaperone of Hsp90, is downregulated in relapsed MM patients, especially after BTZ treatment, suggesting a link between Cdc37 and BTZ resistance. Suppression of Cdc37 or inhibition of Cdc37/Hsp90 association induces plasma cell dedifferentiation, quiescence of MM cells, and BTZ resistance in MM. Furthermore, we discover that Cdc37 expression correlates positively with Xbp1s, a critical transcription factor for plasma cell differentiation in MM samples. Depletion/inhibition of Cdc37 downregulates Xbp1s, while overexpression of Xbp1s in MM cell lines partially rescues plasma immaturation and BTZ resistance. It is suggested that Xbp1s may act as a key downstream effector of Cdc37. Experiments with a mouse model also demonstrate that Cdc37 inhibition promotes plasma cell immaturation, confers BTZ resistance, and increases MM progression in vivo. Together, we identify a critical factor and a new signaling mechanism that regulate plasma cell immaturation and BTZ resistance in MM cells. Our findings may constitute a novel strategy that overcomes BTZ resistance in MM therapy.

Despite the fact that a few novel agents improve the outcome of patients, MM remains incurable. Hence, developing a novel treatment strategy may prove to be promising for the clinical management of MM. Noncoding small RNAs, a cluster of RNAs that do not encode functional proteins, have been underlined that play a pivotal role in the pathogenesis of MM. Our previous study indicated that miR-15a acted as a tumor suppressor, which inhibited the cell proliferation and promoted the apoptosis of MM cells. The level of miR-15a was downregulated in MM cells and correlated with inferior outcome of MM patients. In the present study, we first developed an oligo-single-stranded DNA mimicking the sequence of hsa-miR-15a-5p (OMM-15a) and modified with locked nucleic acid (LNA-15a) to evaluate its anti-MM effects. Our results indicated that the LNA-15a presented an exciting anti-MM effect that showed notable cell growth suppression and apoptosis promotion in MM and other cancer cell lines through downregulating the expression level of target genes BCL-2, VEGF-A, and PHF19. Moreover, LNA-15a treatment significantly improved the anti-MM activity of bortezomib with the synergism effect in OCI-My5 MM cells. In our in vivo study, LNA-15a treatment significantly suppressed the tumor growth, and prolonged the survival of mice compared with the control group. However, our results indicated that the native form of oligo-single-stranded DNA mimic of hsa-miR-15a-5p (OMM-15a) without any modification had no effective inhibition on cell growth, even after increasing the dosage of OMM-15a in the treatment. Altogether, our finding provides the preclinical rationale to support the oligo-single-stranded DNA mimic of hsa-miR-15a with LNA modification, which is a promising tool for the therapy of both MM and other tumors with miR-15a downregulation.

Single-cell analysis is of significant importance in delineate the exact phylogeny of subclonal population and in discovering subtle diversification. So far studies of intratumor heterogeneity and clonal evolution in multiple myeloma (MM) were largely focused at the bulk tumor population level. Here, we performed quantitative multi-gene fluorescence in situ hybridization (QM-FISH) in 129 longitudinal samples of 57 MM patients. All the patients had newly-diagnosed and relapsed paired samples. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. 43 of 57 patients (75.4%) harbored three or four cytogenetic clones at diagnosis. We delineated the phylogeny of subclonal tumor population and derived the evolutionary architecture in each patient.13q deletion and the first 1q gain tended to be earlier cytogenetic alternation, whereas 16q and 17p deletion were acquired later. Patients with clonal stabilization had a significantly improved OS than those with other evolutionary patterns (median OS, 71.2 vs. 39.7 vs. 35.2 vs. 25.5 months, for stable, differential, branching and linear patterns, respectively, p=0.001). Besides, a high degree of consistency and complementarity across QM-FISH and cFISH was observed in evaluation of cytogenetic evolution pattern in MM. In total, at least two time-point cytogenetic evaluations by cFISH were underwent in 188 MM patients. The proportion of patients with high-risk cytogenetic features was 33% at diagnosis and 49% at relapse. The prognostic value of the presence of high-risk aberrations at diagnosis were attenuated over time (HR=1.79, p=0.002 for survival from diagnosis; HR=1.55, p=0.026 for survival from relapse, ). Survival from relapse were greater influenced by the presence of high-risk aberrations at relapse (HR=2.07, figure 5E) rather than present at diagnosis (HR=1.55). The present study investigated the prognostic value of evolution in copy number or clone size of 1q21 gain/amplification during follow-up. The incidence of patients carrying at least three copies of 1q21 was higher after relapse than at diagnosis (69% vs. 55%, p=0.004).Patients were categorized as six groups according to the change patterns in copy number and clone size of 1q21 gain between the two time-point samplings. Patients without 1q21 gain/amplification at both time points (group B) and patients who had obvious decrease in clone size or loss of 1q21 gain at relapse (group A) experienced similar superior outcome (Failure free survival after relapse (2 nd FFS), 18.1 vs. 27.8 months, p=0.469), whereas patients carrying 1q21 gain/amplification at both time points with or without increase (group C,D) in clone size relatively worse survival (2 nd FFS 12.4 and 10.5 months, respectively, p This study shows that QM-FISH is a valuable tool to elucidate the clonal architecture at single cell level. Clonal evolution pattern is of prognostic significance, highlighting the need for repeated cytogenetic evaluation in relapsed MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: The outcomes of patients with multiple myeloma (MM) have improved due to treatment advances. However, some patients still experience rapid progression, multiple drug resistance or recurrent relapse. Tumor-initiating cells, also known as cancer stem cells (CSC) in some cancer types, have been speculated to induce recurrence of the disease. The aim of this study is to infer the identity of myeloma-initiating cells (MICs) utilizing single-cell transcriptome analysis and explore the unique biological characteristics relating with high-risk and drug-resistance. Method: We applied single cell RNA sequencing to fresh bone marrow mononuclear cell samples collecting from 7 healthy donors and 12 newly diagnosed MM patients utilizing 10x Chromium platform. Results: Firstly, we segregated the patients by tumor cell infiltration at single cell resolution and found that Myc pathway was significantly enriched in patients with high tumor burden (HTB). Next, we performed clustering analysis to tumor cells and identified 13 tumor subpopulations in total. Surprisingly, the distribution pattern of tumor subpopulations presented similarity among HTB patients, whereas we did not find the uniform subpopulation composition among the patients with low (LTB) or medium tumor burden (MTB). Via the tumor subpopulation analysis, we clarified the divergence in biological characteristics of these 13 malignant subpopulations. We identified plasmablasts as displaying high expression of B-cell gene signatures (CD19, CD27, MS4A1 and CD79B) and relatively low expression of plasma cell gene signatures (SDC1 and BCMA). Additionally, we also noted that they showed high level of CD24, which has been validated to be the marker gene for MICs. We next examined proliferative capability and utilized the 70 high-risk gene model and 56 drug resistance-related gene model to further distinguish subpopulations with the most malignant gene expression features. Notably, we found that plasmablasts possessed characteristics of high proliferation, drug-resistance and high-risk gene profiling, indicating their role as the root of myeloma, namely MIC subpopulation. Gene enrichment analysis also implicated that Wnt pathway, Notch pathway, stem cell differentiation pathway and Hedgehog pathway were enriched in MIC subpopulation which were associated with the proliferation, migration and drug resistance of MM. Differentially expressed gene (DEG) analysis showed that common driver genes in myeloma, such as CCND2, ITGB7 and CD74, were upregulated in MIC subpopulation comparing with other subpopulations. Conclusion: Our work presents an integral profiling for tumor cells in myeloma at single cell resolution. We uncovered divergence in the distribution of malignant subclusters across patients and distinct heterogeneity in gene expression across malignant subclusters as well. Plasmablasts expressing high level of CD24, CD27 and dim CD138 presented as the MICs with characteristics of higher proliferation, drug-resistance and high-risk gene profiling. Disclosures No relevant conflicts of interest to declare.

Background Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Despite the therapeutic benefit of the proteasome inhibitor (PI) bortezomib, drug resistance still accounts for majority of tumor relapses and cancer-related death in MM patients. Thus, exploring the mechanism underlying BTZ resistance and investigating a novel treatment strategy are urgent requisites for clinical practise. Indirubin-3’-monoxime (Id-3) is an active ingredient of a traditional Chinese medicine. Method In the present study, we firstly investigated Id-3 effects on myeloma treatment in patient primary samples and myeloma cell lines. We also investigated the molecular mechanism of Id-3 treatment on MM cells via in vivo and in vitro study. Results We first reported that Id-3 is an effective cytotoxicity agentia against MM cells growth by inducing the MM cells apoptosis. Id-3 treatment also effectively induces the cell death in bortezomib-resistant MM cell lines and primary myeloma patient samples. Stringently, we found there is a synergistic anti-MM effect between Id-3 and bortezomib. The treatment with lower dosage combination of Id-3 and bortezomib exhibited a notably inhibition of the growth of myeloma cells both in xenograft mouse model and MM-PDX model. Mechanism study revealed that Id-3 treatment efficiently inhibited the chymotrypsin-like and caspase-like activity of proteasome in MM cells. The level of the proteasome 11s activator, PA200 (PSME4) and PA28y (PSME3) was significantly down-regulated both in mRNA and protein level. GEO datasets analysis showed that the high levels of PSME4 and PSME3 were over-expressed in relapsed patients and MM cell lines which correlated with the drug-resistance of MM cells. Kaplan-Meier analysis showed that patients with high-levels of PSME3 or PSME4 had the shortened survival. Knocking-down the level of PSME4 or PSME3 by shRNA noteworthy inhibited the cell growth and enhanced the sensitivity to bortezomib of MM cells. In vivo study also found that down-regulated the level of PSME3 or PSME4 can suppress the proliferation of MM cells and prolong the survival of MM-bearing mouse. Conclusions Taken together, our study supported that Id-3 is an effective agentia against the survival of MM cells both in bortezomib-sensitive and -resistance ones. We further identified that the 11s proteasome activators, PSME3 and PSME4, were down-regulated effectively by Id-3 treatment. High-levels of PSME3 or PSME4 were correlated with the inferior outcome of MM patients. Down-regulating PSME3 and PSME4 by Id-3 or shRNA could efficiently suppressed the growth of MM cells and improve the survival of MM-bearing mice. Our study provides the rational evidence for the clinical application of Id-3 in the treatment of MM.

Introductions Although intratumor heterogeneity and clonal evolution have been inferred in multiple myeloma (MM), this was largely focused at the bulk tumor population level. Single-cell analysis is of significant importance in delineating the exact phylogeny of subclonal population and in discovering subtle diversification. Here, we identified the clonal architecture of different time points using multi-gene fluorescence in situ hybridization (mFISH) at single cell level, and explored the prognostic values of different clonal evolution patterns in MM. Methods We performed mFISH in 129 longitudinal samples of 57 MM patients. All the patients had newly-diagnosed and relapsed paired samples, and 12 patients had cytogenetic evaluation for more than two time points. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. Results 43 of 57 patients (75.4%) harbored three or four cytogenetic clones at diagnosis. We delineated the phylogeny of subclonal tumor population in each patient and established robust trends for the timing of temporal acquisition in the whole cohort using the pairwise precedence. 13q deletion and the first 1q gain tended to be earlier cytogenetic alternation, whereas 16q and 17p deletion were acquired later. The sequence of 13q deletion and 1q21 gain occurrence was identified in 23 patients by the single-cell analysis. 1q21 gain and 13q deletion each occurred first in 12 and 11 patients respectively. Strikingly, patients in whom 13q deletion was acquired first showed a significantly worse survival than 1q21 gain-first patients (median OS 32.9 vs. 71.2 months, p=0.010). We inferred the most likely ancestral relationships between subclones and derived the evolutionary architecture in each patient. Four distinct evolutionary patterns were identified (Figure 1). 18 of 57 (31.6%) patients showed clonal stabilization. These patients were characterized by no novel subclones emerging and no existed subclones disappearing at relapse. Differential evolution was observed in 12 patients, where clonal dynamics resulted from a change in predominant clone from presentation to relapse. The major clone at diagnosis disappeared or decreased to a minor clone while a subclone showed growth advantage and turned to be a major clone at relapse. We found evidence of branching evolution in 9 patients. Here, one or more clones harboring novel cytogenetic abnormalities emerged between the early and late time points, whereas some disappeared. The remainder of patients demonstrated a linear evolution pattern (18/57, 31.6%). The predominant clones acquired one or more novel cytogenetic abnormalities at the later time point. Patients with clonal stabilization had a significantly improved OS than those with other evolutionary patterns (median OS, 71.2 vs. 39.7 vs. 35.2 vs. 25.5 months, for stable, differential, branching and linear patterns, respectively, p=0.001). However, there is no difference in sampling interval among four evolutionary patterns (p=0.131). Therefore, the survival differences were mostly attributable to a significantly shorter failure free survival from relapse (p In order to evaluate the accuracy of abnormalities detection by mFISH, we performed cFISH in these 57 MM patients. Cell fractions of cytogenetic abnormalities detected by mFISH were significantly correlated with that detected by cFISH (p Conclusions These findings suggest that mFISH is a valuable tool for the analysis of clonal phylogeny and evolution pattern of critical cytogenetic aberrations. Patients may benefit from the repeated cytogenetic evaluation, especially for the risk stratification of survival after relapse. Personalized treatment strategy is required for MM patients based on their clonal evolution patterns. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction Mantle cell lymphoma (MCL) is a rare subtype of aggressive non-Hodgkin's lymphoma in which the genomic factors determining the clinical behavior are not fully understood. The genetic heterogeneity in MCL motivates us to characterize the genetic landscape, to define pattern of clonal evolution, and to determine molecular subgroups. Methods We performed whole-exome sequencing on 152 DNA samples derived from 134 MCL patients, which includes 123 untreated and 11 relapsed patients, and 16 with longitudinal samples. 95 patients received high dose cytarabine-based immunochemotherapy from BDH-MCL01 clinical trial (NCT02858804). 48 samples have matched RNA sequencing data for which 42 from untreated and 6 from relapsed patients. We used GATK, MutSig2CV, and GISTIC2.0 to identify driver genetic lesions, applied MutationalPatterns to define mutational signatures, and utilized ABSOLUTE and PhylogicNDT to determine pattern of clonal evolution. We applied NNMF consensus clustering to identify subgroups and evaluated association between genomic features and clinical outcomes. Results The median non-silent mutational burden was 29 per sample (range 8-72). 34 recurrently mutated genes were identified, containing previously reported driver mutations (TP53, ATM, CCND1, KMT2D, NSD2, SMARCA4, ARID1A, NOTCH1, NOTCH2, BIRC3, TRAF2, UBR5) and novel mutations (SP140, SVEP1, LRP1B, LRP2, PCDH10). 7 copy number gain and 13 loss regions were detected as recurrent somatic copy number alterations (frequency>10%, q We defined the clonal status of genetic lesions and pattern of clonal evolution. Del(11q22) and del(9p) tend to be clonal while mutations in NSD2, LRP1B, CTNNA2 were more likely to be subclonal (q 0.5), 4 with modest evolution (0.2 ≤ CCF change ≤ 0.5), 1 without evolution (CCF change < 0.2). Patients with extreme evolution had inferior survival than with those with modest and no evolution (median survival from first sampling was 47.5 months vs not reached, p=0.041, second sampling was 17.1 months vs not reached, p=0.023). We classified MCL into four subsets based on genetic lesions, each with distinct gene expression profile and clinical behavior (Fig C, D). Cluster 1 (C1) and cluster 2-4 (C2-4) were associated with indolent and aggressive types of MCL. Consistent with different cellular origins for two types of MCL, C1 and C2-4 were enriched for gene expression signatures of memory and CCR6 negative light zone B cells, respectively. C1 featured mutated IGHV, CCND1 mutation, amp(11q13) and active BCR signaling. C2 was enriched with del (11q22), del (1p21), ATM mutation and had upregulation of genes involved in the NF-kB and DNA repair pathways while C3 was characterized by mutations in SP140, NOTCH1 and NSD2 and downregulation of gene expression in the NF-kB, BCR signaling, MYC and inflammatory pathways. Interestingly, C4 had the highest incidence of blastoid or pleomorphic MCL (23.7%, p=0.016) and harbored del(17p), del(13q), del(9p) and mutations in TP53 and TRAF2. This cluster carried enrichment of MYC pathway activation and hyperproliferation signatures. These unique gene expression signatures indicated that coordinate genomic factors captured biologic heterogeneity. Importantly, patients in these four clusters had distinct outcomes with median PFS of not reached for C1, 41.2 months for C2, 30.7 months for C3, and 16.1 months for C4 (log rank, p Conclusion Our study provides a portrait of the MCL genetic landscape, uncovers pattern of clonal evolution in MCL, classifies patients with genetic features, links the cluster with gene expression and clinical outcome. The outcome-associated genetic signatures will guide the development of therapies in patients with the greatest need. Figure Disclosures No relevant conflicts of interest to declare.

A large body of evidence indicates that particulate matter (PM)2.5 is associated with various negative effects on human health. However, the impact and molecular mechanism of PM2.5 on the skin have not been elucidated. Therefore, the present study aimed to investigate the effects of two types of PM2.5 [water-soluble extracts (W-PM2.5) and non-water-soluble extracts (NW-PM2.5)] on cell proliferation, cell cycle progression, lipid synthesis, and inflammatory cytokine production of human SZ95 sebocytes. The results demonstrated that NW-PM2.5 and W-PM2.5 exposure dose-dependently inhibited SZ95 sebocyte proliferation by inducing G1 cell arrest. Furthermore, NW-PM2.5 and W-PM2.5 significantly reduced sebaceous lipid synthesis and markedly promoted the production of inflammatory cytokines, including interleukin-1α (IL-1α), IL-6 and IL-8 in SZ95 sebocytes. Additionally, the expression of aryl hydrocarbon (Ah) receptor (AhR), AhR nuclear translocator protein (ARNT), as well as cytochrome P450 1A1 were significantly increased following PM2.5 exposure. Thus, these findings indicate that PM2.5 exerts inhibitory effects on cell proliferation and lipid synthesis, and stimulatory effects on inflammatory cytokine production and AhR signaling activation in human SZ95 sebocytes.