Identification of <i>PSMB4</i> and <i>PSMD4</i> as novel target genes correlated with 1q21 amplification in patients with smoldering myeloma and multiple myeloma

Multiple myeloma (MM) is a malignant plasma cell (PC) dyscrasia characterized by heterogeneous biological features and genetic alterations, resulting in a wide range of disease courses.1,2 Despite all the therapeutic strategies developed in the last three decades, MM is still incurable, and almost all patients will inevitably experience disease progression and eventually relapse.3 Among all the genetic abnormalities, the amplification of the 1q21 region is one of the most frequent cytogenetic abnormalities occurring in malignant PC and it has become a new prognostic factor in MM patients.4,5 The incidence of gain and/or amplification of the 1q21 locus (1q21+) increases with disease progression. It can be detected in around 30-45% of patients with smoldering MM (SMM) and newly diagnosed MM (NDMM), and in around 70% of relapsed/refractory MM patients (RRMM).6 The impact of 1q21 on disease progression at an early stage has not been widely investigated. A few studies have suggested that the acquisition of extra 1q21 copies may play a role in disease progression.7,8 In fact, SMM patients with 1q21+ may be more likely to progress to MM than patients without 1q21+.8 Recent studies have demonstrated that the 1q21 copy number has a different impact on the responsiveness to MM treatments, especially proteasome inhibition (PI).9 PI is a well-established anti-cancer treatment approach used in MM. Throughout the years, the implementation of PI drugs as part of standard MM therapy has continued to improve the quality of life and clinical outcomes of MM patients. Furthermore, additional copies of 1q21 have been associated with PI resistance and recurrence of the disease in patients with 1q21+, limiting the long-term medical utility of PI.9,10 Recent studies have demonstrated that patients with 1q21+ treated with combination treatment with bortezomib (Bor) have inferior progression-free survival and overall survival compared to patients who do not present 1q21+.11 Similar results were observed when patients harboring 1q21 ampliSeveral genes are known to be deregulated upon the amplification of the 1q21 locus;9 nonetheless, the pathogenic and their possible role as druggable targets is not fully understood. In our study, we analyzed primary MM bone marrow (BM) PC from both SMM and NDMM patients to identify gene

Orthogonal Proteogenomic Analysis Identifies the Druggable PA2G4-MYC Axis in 3q26 AML

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML

Targeting DNA2 Overcomes Metabolic Reprogramming in Multiple Myeloma

DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover mechanisms through which MM cells overcome DNA damage, we investigate how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting Interleukin enhancer binding factor 2 (ILF2), a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identify the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells\u27 ability to overcome ILF2 ASO-induced DNA damage, as being essential to counteracting oxidative DNA damage. Our study reveals a mechanism of vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation

Bone marrow mesenchymal stem cells chimerism after allogeneic hematopoietic stem cells transplantation

Le cellule stromali mesenchimali midollari (MSCs) sono cellule primitive e indifferenziate, capaci di autorinnovarsi e di dare origine a differenti linee cellulari. Sono componenti fondamentali del microambiente ematopoietico, composto da una complessa rete di cellule e da una matrice extracellulare che cooperano al fine di regolare il normale processo emopoietico. Le MSCs possono essere isolate dal midollo osseo e producono fattori di crescita e citochine che promuovono l’espansione e la differenziazione delle cellule ematopoietiche. Molti studi hanno messo in evidenza come le MSCs influenzino la reazione al trapianto allogenico e la malattia del trapianto contro l’ospite (GvHD), così come le malattie infiammatorie autoimmuni e altri disordini in cui intervengono i meccanismi di immunomodulazione e di riparazione dei tessuti. Partendo da questa evidenza e dalla possibilità di poter attuare diverse tipologie di trapianto emopoietico nella nostra Unità, abbiamo valutato l’impatto delle MSCs sull’emopoiesi e sull’attecchimento. In questo studio abbiamo analizzato 10 pazienti con neoplasie ematologiche 8 dei quali sottoposti a trapianto aploidentico di cellule staminali da sangue periferico con deplezione TCRαβ/CD19, uno con deplezione CD3/CD19 e Ciclofosfamide post HSCT e un paziente sottoposto a trapianto di midollo da donatore familiare HLA-compatibile. Tutti I pazienti avevano ricevuto l’inoculo da un donatore di sesso opposto. Le MSCs sono state analizzate da campioni di aspirato midollare a differenti time point dopo il trapianto, mediante FISH, utilizzando la sonda CEP X SO/Y SG, specifica per la regione alfa satellite centromerica del cromosoma X e per la regione del DNA satellite III del cromosoma Y. I nostri risultati mostrano che le MSCs rimangono di origine del ricevente in tutti I pazienti sia nelle prime fasi dopo il trapianto che successivamente. I nostri dati confermano i dati già pubblicati in letteratura che dimostrano come l’attecchimento delle MSCs sia un evento estremamente raro e non esista differenza a seconda del tipo di trapianto o fonte di cellule staminali emopoietiche.Abstract Bone marrow-derived mesenchymal stem cells (MSCs) have been defined as primitive, undifferentiated cells, capable of self-renewal and with the ability to give rise to different cell lineages. They are key components of the hematopoietic microenvironment, that is composed of a complex network of cells and extra cellular matrix cooperating to regulate normal hematopoiesis. MSCs can be isolated from bone marrow and they produce growth factors and cytokines that promote hematopoietic cell expansion and differentiation. Several studies have shown that MSCs impact on allograft reaction and graft versus host disease (GvHD), as well as on autoimmune inflammatory disease and other disorders in which immunomodulation and tissue repair are required. Given the different types of hematopoietic transplantation performed in our Bone Marrow Transplant Unit, we evaluated the behavior of MSCs in our setting of transplant, looking at the impact on hematopoiesis and engraftment. In this study we have so far analyzed 10 patients with hematological disease 8 of which received peripheral blood stem cell (PBSC) haploidentical transplantation with T αβ/CD19 depletion, one with CD3/CD19 depletion and Cyclofosfamide post HSCT and one who received bone marrow transplantation from HLA-matching family donor. All patients received grafts from sex mismatched donors. Isolated MSCs were analyzed from sample of bone marrow aspiration at different time point post HSCT by FISH using the CEP X SO/Y SG probe, specific for the alpha satellite centromeric X chromosome region and for the satellite DNA III Y chromosome region. Our results show that MSCs remain of recipient origin in all patients in the early and later stages post transplant confirming that the engraftment of donor MSCs is an extremely rare event, and there is no difference depending on type of transplant or source of hematopoietic stem cells

Role of 1q21 in Multiple Myeloma: From Pathogenesis to Possible Therapeutic Targets

Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) in the bone marrow (BM). The amplification of 1q21 is one of the most common cytogenetic abnormalities occurring in around 40% of de novo patients and 70% of relapsed/refractory MM. Patients with this unfavorable cytogenetic abnormality are considered to be high risk with a poor response to standard therapies. The gene(s) driving amplification of the 1q21 amplicon has not been fully studied. A number of clear candidates are under investigation, and some of them (IL6R, ILF2, MCL-1, CKS1B and BCL9) have been recently proposed to be potential drivers of this region. However, much remains to be learned about the biology of the genes driving the disease progression in MM patients with 1q21 amp. Understanding the mechanisms of these genes is important for the development of effective targeted therapeutic approaches to treat these patients for whom effective therapies are currently lacking. In this paper, we review the current knowledge about the pathological features, the mechanism of 1q21 amplification, and the signal pathway of the most relevant candidate genes that have been suggested as possible therapeutic targets for the 1q21 amplicon

Application of Next-Generation Sequencing for the Genomic Characterization of Patients with Smoldering Myeloma

Genomic analysis could contribute to a better understanding of the biological determinants of the evolution of multiple myeloma (MM) precursor disease and an improved definition of high-risk patients. To assess the feasibility and value of next-generation sequencing approaches in an asymptomatic setting, we performed a targeted gene mutation analysis and a genome-wide assessment of copy number alterations (CNAs) by ultra-low-pass whole genome sequencing (ULP-WGS) in six patients with monoclonal gammopathy of undetermined significance and 25 patients with smoldering MM (SMM). Our comprehensive genomic characterization highlighted heterogeneous but substantial values of the tumor fraction, especially in SMM; a rather high degree of genomic complexity, in terms of both mutations and CNAs, and inter-patient variability; a higher incidence of gene mutations and CNAs in SMM, confirming ongoing evolution; intraclonal heterogeneity; and instances of convergent evolution. ULP-WGS of these patients proved effective in revealing the marked genome-wide level of their CNAs, most of which are not routinely investigated. Finally, the analysis of our small SMM cohort suggested that chr(8p) deletions, the DNA tumor fraction, and the number of alterations may have clinical relevance in the progression to overt MM. Although validation in larger series is mandatory, these findings highlight the promising impact of genomic approaches in the clinical management of SMM

Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties

The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14(+) cells obtained from patients with multiple myeloma or from a human monocytic cell line. LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3(+) cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes. In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity