Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism.

The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.

Update and European consensus on a patient-centered core outcome set for multiple myeloma in clinical practice and research.

Not available.

Predictors of unsustained measurable residual disease negativity in patients with multiple myeloma.

ABSTRACT: The prognostic impact of achieving and in particular maintaining measurable residual disease (MRD) negativity in multiple myeloma is now established; therefore, identifying among MRD-negative patients the ones at higher risk of losing MRD negativity is of importance. We analyzed predictors of unsustained MRD negativity in patients enrolled in the FORTE trial (NCT02203643). MRD was performed by multiparameter flow cytometry (sensitivity of 10-5) at premaintenance and every 6 months thereafter. The cumulative incidence (CI) of MRD resurgence and/or progression was analyzed in MRD-negative patients. A total of 306 of 474 (65%) MRD-negative patients were analyzed. After a median follow-up of 50.4 months from MRD negativity, 185 of 306 (60%) patients were still MRD negative and progression free, 118 (39%) lost their MRD-negative status, and 3 patients (1%) died without progression. Amp1q vs normal (4-year CI, 63% vs 34), ≥2 concomitant high-risk cytogenetic abnormalities vs 0 (4-year CI, 59% vs 33%), circulating tumor cells at baseline (high vs low at 4-year CI, 62% vs 32%), and time-to-reach MRD negativity postconsolidation vs preconsolidation (4-year CI, 46% vs 35%) were associated with a higher risk of unsustained MRD negativity in a multivariate Fine-Gray model. During the first 2 years of maintenance, patients receiving carfilzomib-lenalidomide vs lenalidomide alone had a lower risk of unsustained MRD negativity (4-year CI, 20% vs 33%).

Genomic and immune determinants of resistance to anti-CD38 monoclonal antibody-based therapy in relapsed refractory multiple myeloma.

Anti-CD38 antibody therapies have transformed multiple myeloma (MM) treatment. However, a large fraction of patients inevitably relapses. To understand this, we investigated 32 relapsed MM patients treated with daratumumab, lenalidomide, and dexamethasone (Dara-Rd; NCT03848676 ). Whole genome sequencing (WGS) before and after treatment pinpointed genomic drivers associated with early progression, including RPL5 loss and APOBEC mutagenesis. Flow cytometry on 202 blood samples, collected every three months until progression for 31 patients, revealed distinct immune changes significantly impacting clinical outcomes. Progressing patients exhibited significant depletion of CD38+ NK cells, persistence of T cell exhaustion, and reduced depletion of T-reg cells over time. These findings underscore the influence of immune composition and daratumumab-induced immune changes in promoting MM resistance. Integrating genomics and flow cytometry unveiled associations between adverse genomic features and immune patterns. Overall, this study sheds light on the intricate interplay between genomic complexity and the immune microenvironment driving resistance to Dara-Rd.

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies. METHODS: We performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt's and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment' synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy. RESULTS: We identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects. CONCLUSIONS: The present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Prospective evaluation of minimal residual disease in the phase II FORTE trial: a head-to-head comparison between multiparameter flow cytometry and next-generation sequencing.

Background: Limited data are available on the concordance between multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) for minimal residual disease (MRD) detection in a large trial for multiple myeloma (MM) patients. Methods: MRD was explored in the FORTE trial for transplant-eligible MM patients randomised to three carfilzomib-based induction-intensification-consolidation treatments and carfilzomib-lenalidomide (KR) vs R maintenance. MRD was assessed by 8-colour 2nd-generation flow cytometry in patients with ≥very good partial response before maintenance. NGS was performed in case of suspected complete response (CR) in a correlative subanalysis. Biological/prognostic concordance between MFC and NGS, conversion to MRD negativity during maintenance, and 1-year/2-year sustained MRD negativity were explored. Findings: Between September 28, 2015 and December 22, 2021, 2020 samples were available for MFC and 728 for the simultaneous MFC/NGS correlation in the "suspected CR population". Median follow-up was 62 months. Biological agreement was 87% at the 10-5 and 83% at the 10-6 cut-offs. A remarkable prognostic concordance was observed: hazard ratios in MFC-MRD and NGS-MRD-negative vs -positive patients were 0.29 and 0.27 for progression-free survival (PFS) and 0.35 and 0.31 for overall survival, respectively (p < 0.05). During maintenance, 4-year PFS was 91% and 97% in 1-year sustained MFC-MRD-negative and NGS-MRD-negative patients (10-5), respectively, and 99% and 97% in 2-year sustained MFC-MRD-negative and NGS-MRD-negative patients, regardless of treatment received. The conversion rate from pre-maintenance MRD positivity to negativity during maintenance was significantly higher with KR vs R both by MFC (46% vs 30%, p = 0.046) and NGS (56% vs 30%, p = 0.046). Interpretation: The significant biological/clinical concordance between MFC and NGS at the same sensitivity suggests their possible use in the evaluation of one of the currently strongest predictors of outcome. Funding: Amgen, Celgene/Bristol Myers Squibb, Multiple Myeloma Research Foundation.

Impact of minimal residual disease standardised assessment by FDG-PET/CT in transplant-eligible patients with newly diagnosed multiple myeloma enrolled in the imaging sub-study of the FORTE trial.

Background: 18F-FDG-PET/CT is the current standard technique to define minimal residual disease (MRD) outside the bone marrow (BM) in multiple myeloma (MM), recently standardised applying the Deauville scores (DS) to focal lesions (FS) and bone marrow uptake (BMS) and defining the complete metabolic response (CMR) as uptake below the liver background (DS <4). Methods: In this analysis, we aimed at confirming the role of CMR, and complementarity with BM multiparameter flow cytometry (MFC) at 10-5, in an independent cohort of newly diagnosed transplant-eligible MM patients previously enrolled in the phase II randomised FORTE trial. 109 of the 474 global patients enrolled in the trial between February 23, 2015, and April 5, 2017, who had paired PET/CT (performed at baseline [B] and preceding maintenance therapy [PM]) and MFC evaluation, were included in this analysis. Findings: At B, 93% of patients had focal lesions within the bones (FS ≥4 in 89%) and 99% increased BM uptake (BMS ≥4 in 61%). At PM, CMR was achieved in 63% of patients, which was a strong predictor for prolonged PFS in univariate analysis at landmark time PM (HR 0.40, P = 0.0065) and in Cox multivariate analysis (HR 0.31, P = 0.0023). Regarding OS, a trend in favour of CMR was present in univariate (HR 0.44, P = 0.094), and Cox multivariate model (HR 0.17, P = 0.0037). Patients achieving both PET/CT CMR and MFC negativity at PM showed significantly extended PFS in univariate (HR 0.45, P = 0.020) and multivariate analysis (HR 0.41, P = 0.015). Interpretation: We herein confirm the applicability and validity of DS criteria to define CMR and its prognostic relevance and complementarity with MFC at the BM level. Funding: Amgen, Celgene/Bristol Myers Squibb, Italian Ministry of Health (RC-2022-2773423).

High levels of hypusinated eIF5A in leiomyoma and leiomyosarcoma pathologies: a possible novel therapeutic target.

RESEARCH QUESTION: Is the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis? DESIGN: The hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry. RESULTS: The hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented. CONCLUSIONS: These data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies.

Immune Reconstitution and Vaccinations in Multiple Myeloma: A Report From the 19th International Myeloma Society Annual Workshop.

Given the significance of the immune system and the important role of therapies within the context of the immune system in plasma cell disorders, the International Myeloma Society annual workshop convened a session dedicated to this topic. A panel of experts covered various aspects of immune reconstitution and vaccination. The top oral presentations were highlighted and discussed. This is a report of the proceedings.

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Normalization of the Immunological Microenvironment and Sustained Minimal Residual Disease Negativity: Do We Need Both for Long-Term Control of Multiple Myeloma?

Over the past two decades, the treatment landscape for multiple myeloma (MM) has progressed significantly, with the introduction of several new drug classes that have greatly improved patient outcomes. At present, it is well known how the bone marrow (BM) microenvironment (ME) exerts an immunosuppressive action leading to an exhaustion of the immune system cells and promoting the proliferation and sustenance of tumor plasma cells. Therefore, having drugs that can reconstitute a healthy BM ME can improve results in MM patients. Recent findings clearly demonstrated that achieving minimal residual disease (MRD) negativity and sustaining MRD negativity over time play a pivotal prognostic role. However, despite the achievement of MRD negativity, patients may still relapse. The understanding of immunologic changes in the BM ME during treatment, complemented by a deeper knowledge of plasma cell genomics and biology, will be critical to develop future therapies to sustain MRD negativity over time and possibly achieve an operational cure. In this review, we focus on the components of the BM ME and their role in MM, on the prognostic significance of MRD negativity and, finally, on the relative contribution of tumor plasma cell biology and BM ME to long-term disease control.

Structural-Functional Relationship of the Ribonucleolytic Activity of aIF5A from Sulfolobus solfataricus.

The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, we have undertaken a structural and functional characterization of aIF5A from the crenarchaeal Sulfolobus solfataricus model organism. We confirm the association of aIF5A with several RNA molecules in vivo and demonstrate that the protein is endowed with a ribonuclease activity which is specific for long and structured RNA. By means of biochemical and structural approaches we show that aIF5A can exist in both monomeric and dimeric conformations and the monomer formation is favored by the association with RNA. Finally, modelling of the three-dimensional structure of S. solfataricus aIF5A shows an extended positively charged surface which may explain its strong tendency to associate to RNA in vivo.

Second Revision of the International Staging System (R2-ISS) for Overall Survival in Multiple Myeloma: A European Myeloma Network (EMN) Report Within the HARMONY Project.

PURPOSE: Patients with newly diagnosed multiple myeloma (NDMM) show heterogeneous outcomes, and approximately 60% of them are at intermediate-risk according to the Revised International Staging system (R-ISS), the standard-of-care risk stratification model. Moreover, chromosome 1q gain/amplification (1q+) recently proved to be a poor prognostic factor. In this study, we revised the R-ISS by analyzing the additive value of each single risk feature, including 1q+. PATIENTS AND METHODS: The European Myeloma Network, within the HARMONY project, collected individual data from 10,843 patients with NDMM enrolled in 16 clinical trials. An additive scoring system on the basis of top features predicting progression-free survival (PFS) and overall survival (OS) was developed and validated. RESULTS: In the training set (N = 7,072), at a median follow-up of 75 months, ISS, del(17p), lactate dehydrogenase, t(4;14), and 1q+ had the highest impact on PFS and OS. These variables were all simultaneously present in 2,226 patients. A value was assigned to each risk feature according to their OS impact (ISS-III 1.5, ISS-II 1, del(17p) 1, high lactate dehydrogenase 1, and 1q+ 0.5 points). Patients were stratified into four risk groups according to the total additive score: low (Second Revision of the International Staging System [R2-ISS]-I, 19.2%, 0 points), low-intermediate (II, 30.8%, 0.5-1 points), intermediate-high (III, 41.2%, 1.5-2.5 points), high (IV, 8.8%, 3-5 points). Median OS was not reached versus 109.2 versus 68.5 versus 37.9 months, and median PFS was 68 versus 45.5 versus 30.2 versus 19.9 months, respectively. The score was validated in an independent validation set (N = 3,771, of whom 1,214 were with complete data to calculate R2-ISS) maintaining its prognostic value. CONCLUSION: The R2-ISS is a simple prognostic staging system allowing a better stratification of patients with intermediate-risk NDMM. The additive nature of this score fosters its future implementation with new prognostic variables.

Risk Stratification Before and During Treatment in Newly Diagnosed Multiple Myeloma: From Clinical Trials to the Real-World Setting.

Multiple Myeloma (MM) is a hematologic malignancy characterized by a wide clinical and biological heterogeneity leading to different patient outcomes. Various prognostic tools to stratify newly diagnosed (ND)MM patients into different risk groups have been proposed. At baseline, the standard-of-care prognostic score is the Revised International Staging System (R-ISS), which stratifies patients according to widely available serum markers (i.e., albumin, ß 2-microglobulin, lactate dehydrogenase) and high-risk cytogenetic abnormalities detected by fluorescence in situ hybridization. Though this score clearly identifies a low-risk and a high-risk population, the majority of patients are categorized as at "intermediate risk". Although new prognostic factors identified through molecular assays (e.g., gene expression profiling, next-generation sequencing) are now available and may improve risk stratification, the majority of them need specialized centers and bioinformatic expertise that may preclude their broad application in the real-world setting. In the last years, new tools to monitor response and measurable residual disease (MRD) with very high sensitivity after the start of treatment have been developed. MRD analyses both inside and outside the bone marrow have a strong prognostic impact, and the achievement of MRD negativity may counterbalance the high-risk behavior identified at baseline. All these techniques have been developed in clinical trials. However, their efficient application in real-world clinical practice and their potential role to guide treatment-decision making are still open issues. This mini review will cover currently known prognostic factors identified before and during first-line treatment, with a particular focus on their potential applications in real-world clinical practice.

Immune dysfunctions affecting bone marrow Vγ9Vδ2 T cells in multiple myeloma: Role of immune checkpoints and disease status.

Introduction: Bone marrow (BM) Vγ9Vδ2 T cells are intrinsically predisposed to sense the immune fitness of the tumor microenvironment (TME) in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Methods: In this work, we have used BM Vγ9Vδ2 T cells to interrogate the role of the immune checkpoint/immune checkpoint-ligand (ICP/ICP-L) network in the immune suppressive TME of MM patients. Results: PD-1+ BM MM Vγ9Vδ2 T cells combine phenotypic, functional, and TCR-associated alterations consistent with chronic exhaustion and immune senescence. When challenged by zoledronic acid (ZA) as a surrogate assay to interrogate the reactivity to their natural ligands, BM MM Vγ9Vδ2 T cells further up-regulate PD-1 and TIM-3 and worsen TCR-associated alterations. BM MM Vγ9Vδ2 T cells up-regulate TIM-3 after stimulation with ZA in combination with αPD-1, whereas PD-1 is not up-regulated after ZA stimulation with αTIM-3, indicating a hierarchical regulation of inducible ICP expression. Dual αPD-1/αTIM-3 blockade improves the immune functions of BM Vγ9Vδ2 T cells in MM at diagnosis (MM-dia), whereas single PD-1 blockade is sufficient to rescue BM Vγ9Vδ2 T cells in MM in remission (MM-rem). By contrast, ZA stimulation induces LAG-3 up-regulation in BM Vγ9Vδ2 T cells from MM in relapse (MM-rel) and dual PD-1/LAG-3 blockade is the most effective combination in this setting. Discussion: These data indicate that: 1) inappropriate immune interventions can exacerbate Vγ9Vδ2 T-cell dysfunction 2) ICP blockade should be tailored to the disease status to get the most of its beneficial effect.

Carfilzomib with cyclophosphamide and dexamethasone or lenalidomide and dexamethasone plus autologous transplantation or carfilzomib plus lenalidomide and dexamethasone, followed by maintenance with carfilzomib plus lenalidomide or lenalidomide alone for patients with newly diagnosed multiple myeloma (FORTE): a randomised, open-label, phase 2 trial.

BACKGROUND: Bortezomib-based induction followed by high-dose melphalan (200 mg/m2) and autologous stem-cell transplantation (MEL200-ASCT) and maintenance treatment with lenalidomide alone is the current standard of care for young and fit patients with newly diagnosed multiple myeloma. We aimed to evaluate the efficacy and safety of different carfilzomib-based induction and consolidation approaches with or without transplantation and of maintenance treatment with carfilzomib plus lenalidomide versus lenalidomide alone in newly diagnosed multiple myeloma. METHODS: UNITO-MM-01/FORTE was a randomised, open-label, phase 2 trial done in 42 Italian academic and community practice centres. We enrolled transplant-eligible patients with newly diagnosed multiple myeloma aged 65 years or younger with a Karnofsky Performance Status of 60% or higher. Patients were stratified according to International Staging System stage (I vs II/III) and age (<60 years vs 60-65 years) and randomly assigned (1:1:1) to KRd plus ASCT (four 28-day induction cycles with carfilzomib plus lenalidomide plus dexamethasone [KRd], melphalan at 200 mg/m2 and autologous stem-cell transplantation [MEL200-ASCT], followed by four 28-day KRd consolidation cycles), KRd12 (12 28-day KRd cycles), or KCd plus ASCT (four 28-day induction cycles with carfilzomib plus cyclophosphamide plus dexamethasone [KCd], MEL200-ASCT, and four 28-day KCd consolidation cycles). Carfilzomib 36 mg/m2 was administered intravenously on days 1, 2, 8, 9, 15, and 16; lenalidomide 25 mg administered orally on days 1-21; cyclophosphamide 300 mg/m2 administered orally on days 1, 8, and 15; and dexamethasone 20 mg administered orally or intravenously on days 1, 2, 8, 9, 15, 16, 22, and 23. Thereafter, patients were stratified according to induction-consolidation treatment and randomly assigned (1:1) to maintenance treatment with carfilzomib plus lenalidomide or lenalidomide alone. Carfilzomib 36 mg/m2 was administered intravenously on days 1-2 and 15-16 every 28 days for up to 2 years; lenalidomide 10 mg was administered orally on days 1-21 every 28 days until progression or intolerance in both groups. The primary endpoints were the proportion of patients with at least a very good partial response after induction with KRd versus KCd and progression-free survival with carfilzomib plus lenalidomide versus lenalidomide alone as maintenance treatment, both assessed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02203643. Study recruitment is complete, and all patients are in the follow-up or maintenance phases. FINDINGS: Between Feb 23, 2015, and April 5, 2017, 474 patients were randomly assigned to one of the induction-intensification-consolidation groups (158 to KRd plus ASCT, 157 to KRd12, and 159 to KCd plus ASCT). The median duration of follow-up was 50·9 months (IQR 45·7-55·3) from the first randomisation. 222 (70%) of 315 patients in the KRd group and 84 (53%) of 159 patients in the KCd group had at least a very good partial response after induction (OR 2·14, 95% CI 1·44-3·19, p=0·0002). 356 patients were randomly assigned to maintenance treatment with carfilzomib plus lenalidomide (n=178) or lenalidomide alone (n=178). The median duration of follow-up was 37·3 months (IQR 32·9-41·9) from the second randomisation. 3-year progression-free survival was 75% (95% CI 68-82) with carfilzomib plus lenalidomide versus 65% (58-72) with lenalidomide alone (hazard ratio [HR] 0·64 [95% CI 0·44-0·94], p=0·023). During induction and consolidation, the most common grade 3-4 adverse events were neutropenia (21 [13%] of 158 patients in the KRd plus ASCT group vs 15 [10%] of 156 in the KRd12 group vs 18 [11%] of 159 in the KCd plus ASCT group); dermatological toxicity (nine [6%] vs 12 [8%] vs one [1%]); and hepatic toxicity (13 [8%] vs 12 [8%] vs none). Treatment-related serious adverse events were reported in 18 (11%) of 158 patients in the KRd-ASCT group, 29 (19%) of 156 in the KRd12 group, and 17 (11%) of 159 in the KCd plus ASCT group; the most common serious adverse event was pneumonia, in seven (4%) of 158, four (3%) of 156, and five (3%) of 159 patients. Treatment-emergent deaths were reported in two (1%) of 158 patients in the KRd plus ASCT group, two (1%) of 156 in the KRd12 group, and three (2%) of 159 in the KCd plus ASCT group. During maintenance, the most common grade 3-4 adverse events were neutropenia (35 [20%] of 173 patients on carfilzomib plus lenalidomide vs 41 [23%] of 177 patients on lenalidomide alone); infections (eight [5%] vs 13 [7%]); and vascular events (12 [7%] vs one [1%]). Treatment-related serious adverse events were reported in 24 (14%) of 173 patients on carfilzomib plus lenalidomide versus 15 (8%) of 177 on lenalidomide alone; the most common serious adverse event was pneumonia, in six (3%) of 173 versus five (3%) of 177 patients. One patient died of a treatment-emergent adverse event in the carfilzomib plus lenalidomide group. INTERPRETATION: Our data show that KRd plus ASCT showed superiority in terms of improved responses compared with the other two treatment approaches and support the prospective randomised evaluation of KRd plus ASCT versus standards of care (eg, daratumumab plus bortezomib plus thalidomide plus dexamethasone plus ASCT) in transplant-eligible patients with multiple myeloma. Carfilzomib plus lenalidomide as maintenance therapy also improved progression-free survival compared with the standard-of-care lenalidomide alone. FUNDING: Amgen, Celgene/Bristol Myers Squibb. TRANSLATION: For the Italian translation of the abstract see Supplementary Materials section.

Functional Impact of Genomic Complexity on the Transcriptome of Multiple Myeloma.

PURPOSE: Multiple myeloma is a biologically heterogenous plasma-cell disorder. In this study, we aimed at dissecting the functional impact on transcriptome of gene mutations, copy-number abnormalities (CNA), and chromosomal rearrangements (CR). Moreover, we applied a geno-transcriptomic approach to identify specific biomarkers for personalized treatments. EXPERIMENTAL DESIGN: We analyzed 514 newly diagnosed patients from the IA12 release of the CoMMpass study, accounting for mutations in multiple myeloma driver genes, structural variants, copy-number segments, and raw-transcript counts. We performed an in silico drug sensitivity screen (DSS), interrogating the Cancer Dependency Map (DepMap) dataset after anchoring cell lines to primary tumor samples using the Celligner algorithm. RESULTS: Immunoglobulin translocations, hyperdiploidy and chr(1q)gain/amps were associated with the highest number of deregulated genes. Other CNAs and specific gene mutations had a lower but very distinct impact affecting specific pathways. Many recurrent genes showed a hotspot (HS)-specific effect. The clinical relevance of double-hit multiple myeloma found strong biological bases in our analysis. Biallelic deletions of tumor suppressors and chr(1q)-amplifications showed the greatest impact on gene expression, deregulating pathways related to cell cycle, proliferation, and expression of immunotherapy targets. Moreover, our in silico DSS showed that not only t(11;14) but also chr(1q)gain/amps and CYLD inactivation predicted differential expression of transcripts of the BCL2 axis and response to venetoclax. CONCLUSIONS: The multiple myeloma genomic architecture and transcriptome have a strict connection, led by CNAs and CRs. Gene mutations impacted especially with HS-mutations of oncogenes and biallelic tumor suppressor gene inactivation. Finally, a comprehensive geno-transcriptomic analysis allows the identification of specific deregulated pathways and candidate biomarkers for personalized treatments in multiple myeloma.

Control of the eIF4E activity: structural insights and pharmacological implications.

The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E-eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

Minimal residual disease assessment by multiparameter flow cytometry in transplant-eligible myeloma in the EMN02/HOVON 95 MM trial.

Minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is the most effective tool to define a deep response in multiple myeloma (MM). We conducted an MRD correlative study of the EMN02/HO95 MM phase III trial in newly diagnosed MM patients achieving a suspected complete response before maintenance and every 6 months during maintenance. Patients received high-dose melphalan (HDM) versus bortezomib-melphalan-prednisone (VMP) intensification, followed by bortezomib-lenalidomide-dexamethasone (VRd) versus no consolidation, and lenalidomide maintenance. Bone marrow (BM) samples were processed in three European laboratories, applying EuroFlow-based MFC protocols (eight colors, two tubes) with 10-4-10-5 sensitivity. At enrollment in the MRD correlative study, 76% (244/321) of patients were MRD-negative. In the intention-to-treat analysis, after a median follow-up of 75 months, 5-year progression-free survival was 66% in MRD-negative versus 31% in MRD-positive patients (HR 0.39; p < 0.001), 5-year overall survival was 86% versus 69%, respectively (HR 0.41; p < 0.001). MRD negativity was associated with reduced risk of progression or death in all subgroups, including ISS-III (HR 0.37) and high-risk fluorescence in situ hybridization (FISH) patients (HR 0.38;). In the 1-year maintenance MRD population, 42% of MRD-positive patients at pre-maintenance became MRD-negative after lenalidomide exposure. In conclusion, MRD by MFC is a strong prognostic factor. Lenalidomide maintenance further improved MRD-negativity rate.