Fluorescence in situ hybridization reveals the evolutionary biology of minor clone of gain/amp(1q) in multiple myeloma.

Growing evidence suggests that gain or amplification [gain/amp(1q)] accumulates during disease progression of multiple myeloma (MM). Previous investigations have indicated that small gain/amp(1q) subclones present at the time of diagnosis may evolve into dominant clones upon MM relapse. However, the influence of a minor clone of gain/amp(1q) on MM survival, as well as the correlation between different clonal sizes of gain/amp(1q) and the chromosomal instability (CIN) of MM, remains poorly understood. In this study, we analyzed fluorescence in situ hybridization (FISH) results of 998 newly diagnosed MM (NDMM) patients. 513 patients were detected with gain/amp(1q) at diagnosis. Among these 513 patients, 55 had a minor clone (≤20%) of gain/amp(1q). Patients with a minor clone of gain/amp(1q) displayed similar survival outcomes compared to those without gain/amp(1q). Further analysis demonstrated patients with a minor clone of gain/amp(1q) exhibited a clonal architecture similar to those without gain/amp(1q). Lastly, our results showed a significant increase in the clonal size of the minor clone of gain/amp(1q), frequently observed in MM. These findings suggested that a minor clone of gain/amp(1q) might represent an earlier stage in the pathogenesis of gain/amp(1q) and propose a "two-step" process in the clonal size changes of gain/amp(1q) in MM.

Novel Insights into the Initiation, Evolution, and Progression of Multiple Myeloma by Multi-Omics Investigation.

The evolutionary history of multiple myeloma (MM) includes malignant transformation, followed by progression to pre-malignant stages and overt malignancy, ultimately leading to more aggressive and resistant forms. Over the past decade, large effort has been made to identify the potential therapeutic targets in MM. However, MM remains largely incurable. Most patients experience multiple relapses and inevitably become refractory to treatment. Tumor-initiating cell populations are the postulated population, leading to the recurrent relapses in many hematological malignancies. Clonal evolution of tumor cells in MM has been identified along with the disease progression. As a consequence of different responses to the treatment of heterogeneous MM cell clones, the more aggressive populations survive and evolve. In addition, the tumor microenvironment is a complex ecosystem which plays multifaceted roles in supporting tumor cell evolution. Emerging multi-omics research at single-cell resolution permits an integrative and comprehensive profiling of the tumor cells and microenvironment, deepening the understanding of biological features of MM. In this review, we intend to discuss the novel insights into tumor cell initiation, clonal evolution, drug resistance, and tumor microenvironment in MM, as revealed by emerging multi-omics investigations. These data suggest a promising strategy to unravel the pivotal mechanisms of MM progression and enable the improvement in treatment, both holistically and precisely.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy.

BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. METHODS: The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. FINDINGS: High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. In vitro and in vivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. INTERPRETATION: Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. FUNDING: A full list of funding sources can be found in the acknowledgements section.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma by Targeted Tracking Serum M-Protein Using Mass Spectrometry (EasyM).

PURPOSE: We investigated both the clinical utilities and the prognostic impacts of the clonotypic peptide mass spectrometry (MS)-EasyM, a blood-based minimal residual disease (MRD) monitoring protocol in multiple myeloma. EXPERIMENTAL DESIGN: A total of 447 sequential serum samples from 56 patients with multiple myeloma were analyzed using EasyM. Patient-specific M-protein peptides were sequenced from diagnostic samples; sequential samples were quantified by EasyM to monitor the M-protein. The performance of EasyM was compared with serum immunofixation electrophoresis (IFE), bone marrow multiparameter flow cytometry (MFC), and next-generation flow cytometry (NGF) detection. The optimal balance of EasyM sensitivity/specificity versus NGF (10-5 sensitivity) was determined and the prognostic impact of MS-MRD status was investigated. RESULTS: Of the 447 serum samples detected and measured by EasyM, 397, 126, and 92 had time-matching results for comparison with serum IFE, MFC-MRD, and NGF-MRD, respectively. Using a dotp >0.9 as the MS-MRD positive, sensitivity was 99.6% versus IFE and 100.0% versus MFC and NGF. Using an MS negative cutoff informed by ROC analysis (<1.86% of that at diagnosis), EasyM sensitivity remained high versus IFE (88.3%), MFC (85.1%), and NGF (93.2%), whereas specificity increased to 90.4%, 55.8%, and 93.2%, respectively. In the multivariate analysis, older diagnostic age was an independent predictor for progression-free survival [PFS; high risk (HR), 3.15; 1.26-7.86], the best MS-MRD status (MS-MRD negative) was independent predictor for both PFS (HR, 0.25; 0.12-0.52) and overall survival (HR, 0.16; 0.06-0.40). CONCLUSIONS: EasyM is a highly sensitive and minimal invasive method of MRD monitoring in multiple myeloma; MS-MRD had significant predictive ability for survival outcomes.

Early relapse within 18 months is a powerful dynamic predictor for prognosis and could revise static risk distribution in multiple myeloma.

BACKGROUND: The duration of response to treatment is a major prognostic factor, and early relapse (ER) strongly predicts inferior survival in multiple myeloma (MM). However, the definitions of ER in MM vary from study to study and how to dynamically integrate risk distribution is still unsolved. METHODS: This study evaluated these ER definitions and further investigated the underlying relationship with static risk distribution in 629 newly diagnosed MM (NDMM) patients from the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199). RESULTS: These data indicated that early relapse within 18 months (ER18) after initial treatment was the best time point for identifying early progression and dynamic high-risk in MM. The ER18 population (114 of 587, 19.4%) presented with more aggressive biologic features and the inferior response to treatment compared to a reference cohort (p < .001), with a significantly short median overall survival (OS) of 28.9 months. Multivariate analyses confirmed the most significant prognostic value of ER18 on OS in the context of International Staging System stage, elevated lactate dehydrogenase, thrombocytopenia, cytogenetic abnormalities, and treatment (hazard ratio, 4.467; p < .001). The authors also described the specific transitions from static risk profile to dynamic risk distribution and then constructed a mixed-risk-pattern to identify four novel populations with distinct survival (p < .001). Additionally, the authors proposed a second-state model that predicts dynamic risk changes, enabling a complementary role to the Revised International Staging System model in facilitating individualized systematic treatment. CONCLUSIONS: Collectively, this study concludes that ER18 is a simple and dynamic prognostic predictor in MM. In addition to static risk assessment, dynamic risk plays an important role in survival prediction.

[Establishment of Patient-Derived Xenograft (PDX) Zebrafish Model of Multiple Myeloma and Its Application in Drug Screening].

OBJECTIVE: To establish a MM patient-derived tumor xenograft model (MM-PDX) in zebrafish, and to evaluate the anti-myeloma activity of indirubin-3'-monoxime(I3MO) using this model. METHODS: Zebrafish embryos 2 days after fertilization were transplanted with fluorescence labeled myeloma primary tumor cells, the survival of primary tumor cells in zebrafish was observed at 0,16 and 24 hours after cell injection. The zebrafish embryos after tumor cell transplantation were randomly divided into control group, BTZ treatment and I3MO treatment group. Before and 24 hours after treatment with BTZ and I3MO, the positive area with calcein or Dil in zebrafish were observed under fluorescence microscope to reflect the survival of tumor cells, and it was verified. RESULTS: MM patient derived tumor cells survived in zebrafish. The construction of MM-PDX was successful. Compared with control group, the fluo- rescence area of the BTZ and I3MO treatment groups in zebrafish were significantly decreased(P<0.05), and BTZ and I3MO significantly inhibited the survival of MM cells in zebrafish. CONCLUSION: MM-PDX model was successfully established. Zebrafish model derived from tumor cells of MM patients can be used as a tool for drug screening of MM.

The age-dependent changes in risk weights of the prognostic factors for multiple myeloma.

OBJECTIVE: Multiple myeloma is a highly heterogenous plasma cell malignancy, commonly seen in older patients. Age is one of the important prognostic factors. However, nearly all the prognostic staging systems are based on clinical trials, where patients were relatively fit and young. It is unknown how the presence of biochemical or cytogenetic prognostic factors and their risk weights changes with older age. To further investigate this question, we retrospectively analyzed the data from a consecutive cohort of patients treated with either bortezomib or thalidomide-based therapy. METHODS: This retrospective study was carried out on a cohort of 1125 newly diagnosed multiple myeloma patients, from January 2008 to December 2019. Patients received bortezomib or thalidomide-based induction and maintenance therapy. Patients accepted hematopoietic stem cell transplantation if eligible. Statistical analysis was conducted by Stata/MP 16.0 and SPSS 26.0. RESULTS: With age increasing, the proportion of patients with ISS 3, performance status score ≥2, and the incidence rate of gain(1q) significantly increased. We also found that ISS became less important in older patients. However, cytogenetic abnormalities exerted a consistently adverse impact on survival, both in young and old patients. Older patients had an inferior outcome than their young counterparts. All patients in our cohort benefitted more from bortezomib than thalidomide-based induction therapy, except for patients ≥71 years old. CONCLUSIONS: ISS may lose prognostic value in patients ≥71 years old. Older patients had an inferior outcome and needed more effective and less toxic treatment.Plain Language SummaryMultiple myeloma is a type of blood cancer commonly seen in older people. To treat this disease, genetic abnormality, the poor physical status of patients and the abundance of tumor cells are the main difficulties. We often draw these conclusions from clinical trials. However, clinical trials always enrolled relatively younger patients, so the presence and significance of these factors may vary from clinical trials to the real world. We conducted the study to find out the real risk in both young and old patients. We found that older patients were more likely to have anemia, poor nutritional status and renal function. We also found older patients had more risk of relapse, progression or death than young patients. Frail physical status is the key obstacle to treating older patients, and tumor burden no longer impacts the outcome of these people. Bortezomib is a powerful drug to treat this disease, but patients ≥71 years old had less benefit than younger ones. More studies should focus on older or frail patients as these patients need more effective and less toxic treatment.

Exosome miRNAs profiling in serum and prognostic evaluation in patients with multiple myeloma.

MicroRNAs (MiRNAs) carried by exosomes play pivotal roles in the crosstalk between cell components in the tumor microenvironment. Our study aimed at identifying the expression profile of exosomal miRNAs (exo-miRNAs) in the serum of multiple myeloma (MM) patients and investigating the regulation networks and their potential functions by integrated bioinformatics analysis. Exosomes in serum from 19 newly diagnosed MM patients and 9 healthy donors were isolated and the miRNA profile was investigated by small RNA sequencing. Differential expression of exo-miRNAs was calculated and target genes of miRNAs were predicted. CytoHubba was applied to identify the hub miRNAs and core target genes. The LASSO Cox regression model was used to develop the prognostic model, and the ESTIMATE immune score was calculated to investigate the correlation between the model and immune status in MM patients. The top six hub differentially expressed serum exo-miRNAs were identified. 513 target genes of the six hub exo-miRNAs were confirmed to be differentially expressed in MM cells in the Zhan Myeloma microarray dataset. Functional enrichment analysis indicated that these target genes were mainly involved in mRNA splicing, cellular response to stress, and deubiquitination. 13 core exo-miRNA target genes were applied to create a novel prognostic signature to provide risk stratification for MM patients, which is associated with the immune microenvironment of MM patients. Our study comprehensively investigated the exo-miRNA profiles in MM patients. A novel prognostic signature was constructed to facilitate the risk stratification of MM patients with distinct outcomes.

More accurate prediction of treatment response than mean apparent diffusion coefficient value in multiple myeloma using whole body MRI histogram analysis.

Aim: To assess baseline histogram parameters from apparent diffusion coefficient (ADC) images in predicting early treatment response in newly diagnosed multiple myeloma (NDMM) patients. Methods: The histogram parameters of lesions in 68 NDMM patients were obtained with the Firevoxel software. The presence of deep response after two cycles of induction was recorded. Results: Some parameters were significantly different between the two groups, for example, ADC 75% in lumbar spine (p = 0.026). No significant difference in mean ADC for any anatomic site was found (all p > 0.05). The combination of ADC 75, ADC 90 and ADC 95% in lumbar spine; ADC skewness and ADC kurtosis in rib achieved a sensitivity of 100% in predicting deep response. Conclusion: Histogram analysis of ADC images can describe NDMM heterogeneity and accurately predict treatment response.

Aberrant metabolic processes promote the immunosuppressive microenvironment in multiple myeloma.

Introduction: Multiple myeloma (MM) is still an incurable plasma cell malignancy. The efficacy of immunotherapy on MM remains unsatisfactory, and the underlying molecular mechanisms still are not fully understood. Methods: In this study, we delineated the dynamic features of immune cell in MM bone marrow (BM) along with elevated tumor cell infiltration by single-cell RNA sequencing (scRNA-seq), and investigated the underlying mechanisms on dysfunction of immune cells associated with myelomagenesis. Results: We found that immune cells were activated in those patients with low infiltration of tumor cells, meanwhile suppressed with elevated infiltration of MM cells, which facilitated MM escaping from immune surveillance. Besides PD-1, abnormal expression of PIM kinases, KLRB1 and KLRC1 were involved in the defect of immune cells in MM patients. Importantly, we found aberrant metabolic processes were associated with the immunosuppressive microenvironment in MM patients. Disordered amino acid metabolism promoted the dysfunction of cytotoxicity CD8 T cells as well as lipid metabolism disorder was associated with the dysregulation of NK and DCs in MM. As metabolic checkpoints, PIM kinases would be potential effective strategies for MM immunotherapy. Discussion: In summary, redressing the disordered metabolism should be the key points to get promising effects in immune-based therapies.

Single-cell profiles reveal tumor cell heterogeneity and immunosuppressive microenvironment in Waldenström macroglobulinemia.

BACKGROUND: Waldenström macroglobulinemia (WM) is a rare and incurable indolent B-cell malignancy. The molecular pathogenesis and the role of immunosuppressive microenvironment in WM development are still incompletely understood. METHODS: The multicellular ecosystem in bone marrow (BM) of WM were delineated by single-cell RNA-sequencing (scRNA-seq) and investigated the underlying molecular characteristics. RESULTS: Our data uncovered the heterogeneity of malignant cells in WM, and investigated the kinetic co-evolution of WM and immune cells, which played pivotal roles in disease development and progression. Two novel subpopulations of malignant cells, CD19+CD3+ and CD138+CD3+, co-expressing T-cell marker genes were identified at single-cell resolution. Pseudotime-ordered analysis elucidated that CD19+CD3+ malignant cells presented at an early stage of WM-B cell differentiation. Colony formation assay further identified that CD19+CD3+ malignant cells acted as potential WM precursors. Based on the findings of T cell marker aberrant expressed on WM tumor cells, we speculate the long-time activation of tumor antigen-induced immunosuppressive microenvironment that is involved in the pathogenesis of WM. Therefore, our study further investigated the possible molecular mechanism of immune cell dysfunction. A precursor exhausted CD8-T cells and functional deletion of NK cells were identified in WM, and CD47 would be a potential therapeutic target to reverse the dysfunction of immune cells. CONCLUSIONS: Our study facilitates further understanding of the biological heterogeneity of tumor cells and immunosuppressive microenvironment in WM. These data may have implications for the development of novel immunotherapies, such as targeting pre-exhausted CD8-T cells in WM.

Fluoxetine modifies circadian rhythm by reducing melatonin content in zebrafish.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), increases the serotonin levels in the brain to treat depression. Antidepressants have been demonstrated to modulate circadian rhythm, but the underlying mechanisms by which antidepressants regulate circadian rhythm require more research. This study aimed to investigate the role of FLX on circadian rhythm by analyzing the movement behavior and internal circadian oscillations in zebrafish. The results showed that the expression of clock genes clock1a and bmal1b was significantly down-regulated, and the amplitude reduction and phase shift were observed after FLX treatment. Furthermore, FLX exposure inhibited the expression of aanat2, which led to a decrease in nocturnal melatonin secretion. aanat2-/- larvae showed disrupted circadian rhythm. These findings may help reveal the effect of FLX exposure on the circadian rhythm and locomotor activity. It may provide theoretical data for the clinical application of FLX.

The clinical characteristics and prognosis of patients with primary plasma cell leukemia (pPCL) according to the new IMWG definition criteria.

Recently, the definition of primary plasma cell leukemia (pPCL) has been revised as the presence of circulating plasma cells (CPCs) ≥5% in peripheral blood smear. Consequently, the clinical features and prognosis of this aggressive disease can be truly identified by the larger patient cohort. Herein, we identified 158 new-defined pPCL patients among 2,266 MM patients (7.0%), and such prevalence doubled the previous estimate. Our study firstly provided solid support for the application of the new definition. We also found that cytopenias and adverse prognostic biomarkers were more common in new-defined pPCL compared with MM (p < 0.05). Besides, the patients receiving proteasome inhibitors based regimen in combination with stem cell transplantation could experience a considerable survival benefit. Strikingly, we showed that the presence of conventional high-risk cytogenetic abnormalities in pPCL didn't exert a great prognostic effect like MM, while elevated LDH reflecting tumor cells proliferation rate was the only independent predictor.

Indirubin-3'-monoxime acts as proteasome inhibitor: Therapeutic application in multiple myeloma.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: Altogether, our study indicates that I3MO is agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM. FUNDINGS: A full list of funding can be found in the acknowledgements.

Prediction of Early Treatment Response in Multiple Myeloma Using MY-RADS Total Burden Score, ADC, and Fat Fraction From Whole-Body MRI: Impact of Anemia on Predictive Performance.

BACKGROUND. The recently released Myeloma Response Assessment and Diagnosis System (MY-RADS) for multiple myeloma (MM) evaluation using whole-body MRI (WB-MRI) describes the total burden score. However, assessment is confounded by red bone marrow hyperplasia in anemia. OBJECTIVE. The purpose of this study is to assess the utility of the MY-RADS total burden score, ADC, and fat fraction (FF) from WB-MRI in predicting early treatment response in patients with newly diagnosed MM and to compare the utility of these measures between patients with and without anemia. METHODS. This retrospective study included 56 patients (40 men, 16 women; mean age, 57.4 ± 9.6 [SD] years) with newly diagnosed MM who underwent baseline WB-MRI including DWI and modified Dixon sequences. Two radiologists recorded total burden score using MY-RADS and measured the ADC and FF of diffuse and focal disease sites. Mean values across sites were derived. Interobserver agreement was evaluated, and the mean assessments of the readers were used for further analyses. Presence of deep response after four cycles of induction chemotherapy was recorded. Patients were classified as having anemia if their hemoglobin level was less than 100 g/L. The utility of WBMRI parameters in predicting deep response was assessed. RESULTS. A total of 24 of 56 patients showed deep response, and 25 of 56 patients had anemia. Interobserver agreement, which was expressed using intraclass correlation coefficients, ranged from 0.95 to 0.99. Among patients without anemia, those with deep response compared with those without deep response had a lower total burden score (9.0 vs 18.0), a lower ADC (0.79 × 10-3 mm2/s vs 1.08 × 10-3 mm2/s), and a higher FF (0.21 vs 0.10) (all p < .001). The combination of these three parameters (optimal cutoffs: ≤ 15 for total burden score, ≤ 0.84 × 10-3 mm2/s for ADC, and > 0.16 for FF) achieved sensitivity of 93.8%, specificity of 93.3%, and accuracy of 93.5% for predicting deep response. In patients with anemia, none of the three parameters were significantly different between patients with and without deep response (all p > .05), and the combination of parameters achieved sensitivity of 56.3%, specificity of 100.0%, and accuracy of 72.0%. CONCLUSION. Low total burden score, low ADC, and high FF from WB-MRI may predict deep response in patients with MM, although only among those without anemia. CLINICAL IMPACT. WB-MRI findings may help guide determination of prognosis and initial treatment selection in MM.

Clonal phylogeny and evolution of critical cytogenetic aberrations in multiple myeloma at single-cell level by QM-FISH.

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.

Genomic and transcriptomic profiling reveals distinct molecular subsets associated with outcomes in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a phenotypically and genetically heterogeneous malignancy in which the genetic alterations determining clinical indications are not fully understood. Here, we performed a comprehensive whole-exome sequencing analysis of 152 primary samples derived from 134 MCL patients, including longitudinal samples from 16 patients and matched RNA-Seq data from 48 samples. We classified MCL into 4 robust clusters (C1-C4). C1 featured mutated immunoglobulin heavy variable (IGHV), CCND1 mutation, amp(11q13), and active B cell receptor (BCR) signaling. C2 was enriched with del(11q)/ATM mutations and upregulation of NF-κB and DNA repair pathways. C3 was characterized by mutations in SP140, NOTCH1, and NSD2, with downregulation of BCR signaling and MYC targets. C4 harbored del(17p)/TP53 mutations, del(13q), and del(9p), and active MYC pathway and hyperproliferation signatures. Patients in these 4 clusters had distinct outcomes (5-year overall survival [OS] rates for C1-C4 were 100%, 56.7%, 48.7%, and 14.2%, respectively). We also inferred the temporal order of genetic events and studied clonal evolution of 16 patients before treatment and at progression/relapse. Eleven of these samples showed drastic clonal evolution that was associated with inferior survival, while the other samples showed modest or no evolution. Our study thus identifies genetic subsets that clinically define this malignancy and delineates clonal evolution patterns and their impact on clinical outcomes.

Four and a Half LIM Domains Protein 2 Mediates Bortezomib-Induced Osteogenic Differentiation of Mesenchymal Stem Cells in Multiple Myeloma Through p53 Signaling and ß-Catenin Nuclear Enrichment.

Myeloma bone disease (MBD), caused by the inhibition of osteoblast activity and the activation of osteoclast in the bone marrow environment, is the most frequent and life-threatening complication in multiple myeloma (MM) patients. Bortezomib (Bzb) was shown to promote MM-derived mesenchymal stem cells (MM-MSCs) differentiation to osteoblast in vitro and in animal models, promoting the bone formation and regeneration, may be mediated via ß-catenin/T-cell factor (TCF) pathway. Further defining molecular mechanism of Bzb-enhanced bone formation in MM will be beneficial for the treatment of myeloma patients. The present study has identified for the first time four and a half LIM domains protein 2 (FHL2), a tissue-specific coregulator that interacts with many osteogenic marker molecules, as a therapeutic target to ameliorate MM bone disease. First, increased messenger RNA (mRNA) and protein levels of FHL2, and the mRNA level of main osteoblast markers (including Runx2, ALP, and Col1A1), were found in MM-patients-derived MSCs after Bzb treatment. FHL2 KD with short hairpin RNA (shRNA) reduced the expression of osteoblast marker genes and blocked the osteogenic differentiation of MM-MSCs regardless of the presence or absence of Bzb, implying that FHL2 is an important activator of the osteogenic differentiation of human MSCs under a proteasome inhibition condition. Molecular analysis showed that the enhanced expression of FHL2 was associated with the Bzb-induced upregulation of p53. No significant change at protein level of total ß-catenin was observed with or without Bzb treatment. However, it was mostly enriched to nuclei in MSCs after Bzb treatment. Moreover, ß-catenin was restricted to the perinuclear region in FHL2 KD cells. These data provide evidence that FHL2 is essential for promoting ß-catenin nuclear enrichment in MM-MSCs. In conclusion, FHL2 is critical for Bzb-induced osteoblast differentiation of MM-MSCs and promotes the osteogenesis, through p53 signaling and ß-catenin activation. Targeting FHL2 in MM may provide a new therapeutic strategy for treating MBD.