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' 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.

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 novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting 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 an adaptive metabolic rewiring and rely on oxidative phosphorylation to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identified the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells' ability to overcome ILF2 ASO-induced DNA damage, as being essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration. Our study revealed a novel vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation. STATEMENT OF SIGNIFICANCE: Metabolic reprogramming is a mechanism through which cancer cells maintain survival and become resistant to DNA-damaging therapy. Here, we show that targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.

Molecular Features of the Mesenchymal and Osteoblastic Cells in Multiple Myeloma.

Multiple myeloma (MM) is a monoclonal gammopathy characterized by biological heterogeneity and unregulated proliferation of plasma cells (PCs) in bone marrow (BM). MM is a multistep process based on genomic instability, epigenetic dysregulation and a tight cross-talk with the BM microenvironment that plays a pivotal role supporting the proliferation, survival, drug-resistance and homing of PCs. The BM microenvironment consists of a hematopoietic and a non-hematopoietic compartment, which cooperate to create a tumor environment. Among the non-hematopoietic component, mesenchymal stromal cells (MSCs) and osteoblasts (OBs) appear transcriptionally and functionally different in MM patients compared to healthy donors (HDs) and to patients with pre-malignant monoclonal gammopathies. Alterations of both MSCs and OBs underly the osteolytic lesions that characterize myeloma-associated bone disease. In this review, we will discuss the different characteristics of MSCs and OBs in MM patients, analyzing the transcriptome, the deregulated molecular pathways and the role performed by miRNAs and exosome in the pathophysiology of MM.

Metabolic features of myeloma cells in the context of bone microenvironment: Implication for the pathophysiology and clinic of myeloma bone disease.

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasma cells (PCs) into the bone marrow (BM). The complex interaction between the BM microenvironment and MM PCs can lead to severe impairment of bone remodeling. Indeed, the BM microenvironment exerts a critical role in the survival of malignant PCs. Growing evidence indicates that MM cells have several metabolic features including enhanced glycolysis and an increase in lactate production through the upregulation of glucose transporters and enzymes. More recently, it has been reported that MM cells arehighly glutamine addicted. Interestingly, these metabolic changes in MM cells may affect BM microenvironment cells by altering the differentiation process of osteoblasts from mesenchymal stromal cells. The identification of glutamine metabolism alterations in MM cells and bone microenvironment may provide a rationale to design new therapeutic approaches and diagnostic tools. The osteolytic lesions are the most frequent clinical features in MM patients, often characterized by pathological fractures and acute pain. The use of the newer imaging techniques such as Magnetic Resonance Imaging (MRI) and combined Positron Emission Tomography (PET) and Computerized Tomography (CT) has been introduced into clinical practice to better define the skeletal involvement. Currently, the PET/CT with 18F-fluorodeoxyglucose (FDG) is the diagnostic gold standard to detect active MM bone disease due to the high glycolytic activity of MM cells. However, new tracers are actively under investigation because a portion of MM patients remains negative at the skeletal level by 18F-FDG. In this review, we will summarize the existing knowledge on the metabolic alterations of MM cells considering their impact on the BM microenvironment cells and particularly in the subsequent formation of osteolytic bone lesions. Based on this, we will discuss the identification of possible new druggable targets and the use of novel metabolic targets for PET imaging in the detection of skeletal lesions, in the staging and treatment response of MM patients.

A personalized molecular approach in multiple myeloma: the possible use of RAF/RAS/MEK/ERK and BCL-2 inhibitors.

Multiple myeloma (MM) is a blood cancer that derives from plasma cells (PCs), which will accumulate in the bone marrow (BM). Over time, several drugs have been developed to treat this disease that is still uncurable. The therapies used to treat the disease target immune activity, inhibit proteasome activity, and involve the use of monoclonal antibodies. However, MM is a highly heterogeneous disease, in fact, there are several mutations in signaling pathways that are particularly important for MM cell biology and that are possible therapeutic targets. Indeed, some studies suggest that MM is driven by mutations within the rat sarcoma virus (RAS) signaling cascade, which regulates cell survival and proliferation. The RAS/proto-oncogene, serine/threonine kinase (RAF)/mitogen-activated extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway is deregulated in several cancers, for which drugs have been developed to inhibit these pathways. In addition to the signaling pathways, the disease implements mechanisms to ensure the survival and consequently a high replicative capacity. This strategy consists in the deregulation of apoptosis. In particular, some cases of MM show overexpression of anti-apoptotic proteins belonging to the B cell lymphoma 2 (BCL-2) family that represent a possible druggable target. Venetoclax is an anti-BCL-2 molecule used in hematological malignancies that may be used in selected MM patients based on their molecular profile. We focused on the possible effects in MM of off-label drugs that are currently used for other cancers with the same molecular characteristics. Their use, combined with the current treatments, could be a good strategy against MM.

Immune response to SARS-CoV-2 mRNA vaccination and booster dose in patients with multiple myeloma and monoclonal gammopathies: impact of Omicron variant on the humoral response.

The humoral and cellular response to SARS-CoV-2 mRNA full vaccination and booster dose as well as the impact of the spike variants, including Omicron, are still unclear in patients with multiple myeloma (MM) and those with pre-malignant monoclonal gammopathies. In this study, involving 40 patients, we found that MM patients with relapsed-refractory disease (MMR) had reduced spike-specific antibody levels and neutralizing titers after SARS-CoV-2 vaccination. The five analyzed variants, remarkably Omicron, had a significant negative impact on the neutralizing ability of the vaccine-induced antibodies in all patients with MM and smoldering MM. Moreover, lower spike-specific IL-2-producing CD4+ T cells and reduced cytotoxic spike-specific IFN-γ and TNF-α-producing CD8+ T cells were found in MM patients as compared to patients with monoclonal gammopathy of undetermined significance. We found that a heterologous booster immunization improved SARS-CoV-2 spike humoral and cellular responses in newly diagnosed MM (MMD) patients and in most, but not all, MMR patients. After the booster dose, a significant increase of the neutralizing antibody titers against almost all the analyzed variants was achieved in MMD. However, in MMR patients, Omicron retained a negative impact on neutralizing ability, suggesting further approaches to potentiating the effectiveness of SARS-CoV-2 vaccination in these patients.

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.

Mechanisms of Action of the New Antibodies in Use in Multiple Myeloma.

Monoclonal antibodies (mAbs) directed against antigen-specific of multiple myeloma (MM) cells have Fc-dependent immune effector mechanisms, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP), but the choice of the antigen is crucial for the development of effective immuno-therapy in MM. Recently new immunotherapeutic options in MM patients have been developed against different myeloma-related antigens as drug conjugate-antibody, bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)-T cells. In this review, we will highlight the mechanism of action of immuno-therapy currently available in clinical practice to target CD38, SLAMF7, and BCMA, focusing on the biological role of the targets and on mechanisms of actions of the different immunotherapeutic approaches underlying their advantages and disadvantages with critical review of the literature data.

PD-L1/PD-1 Axis in Multiple Myeloma Microenvironment and a Possible Link with CD38-Mediated Immune-Suppression.

The emerging role of the PD-1/PD-L1 axis in MM immune-microenvironment has been highlighted by several studies. However, discordant data have been reported on PD-1/PD-L1 distribution within the bone marrow (BM) microenvironment of patients with monoclonal gammopathies. In addition, the efficacy of PD-1/PD-L1 blockade as a therapeutic strategy to reverse myeloma immune suppression and inhibit myeloma cell survival still remains unknown. Recent data suggest that, among the potential mechanisms behind the lack of responsiveness or resistance to anti-PD-L1/PD-1 antibodies, the CD38 metabolic pathways involving the immune-suppressive factor, adenosine, could play an important role. This review summarizes the available data on PD-1/PD-L1 expression in patients with MM, reporting the main mechanisms of regulation of PD-1/PD-L1 axis. The possible link between the CD38 and PD-1/PD-L1 pathways is also reported, highlighting the rationale for the potential use of a combined therapeutic approach with CD38 blocking agents and anti-PD-1/PD-L1 antibodies in order to improve their anti-tumoral effect in MM patients.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Novel Approaches to Improve Myeloma Cell Killing by Monoclonal Antibodies.

The monoclonal antibodies (mAbs) have significantly changed the treatment of multiple myeloma (MM) patients. However, despite their introduction, MM remains an incurable disease. The mAbs currently used for MM treatment were developed with different mechanisms of action able to target antigens, such as cluster of differentiation 38 (CD38) and SLAM family member 7 (SLAMF7) expressed by both, MM cells and the immune microenvironment cells. In this review, we focused on the mechanisms of action of the main mAbs approved for the therapy of MM, and on the possible novel approaches to improve MM cell killing by mAbs. Actually, the combination of anti-CD38 or anti-SLAMF7 mAbs with the immunomodulatory drugs significantly improved the clinical effect in MM patients. On the other hand, pre-clinical evidence indicates that different approaches may increase the efficacy of mAbs. The use of trans-retinoic acid, the cyclophosphamide or the combination of anti-CD47 and anti-CD137 mAbs have given the rationale to design these types of combinations therapies in MM patients in the future. In conclusion, a better understanding of the mechanism of action of the mAbs will allow us to develop novel therapeutic approaches to improve their response rate and to overcome their resistance in MM patients.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy.

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

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.

CD14+ CD16+ monocytes are involved in daratumumab-mediated myeloma cells killing and in anti-CD47 therapeutic strategy.

A deep elucidation of the mechanisms of action of anti-CD38 monoclonal antibodies (mAbs), such as daratumumab (DARA), is required to identify patients with multiple myeloma (MM) who are more responsive to this treatment. In the present study, an autologous ex vivo approach was established, focussing on the role of the monocytes in the anti CD38-mediated killing of MM cells. In bone marrow (BM) samples from 29 patients with MM, we found that the ratio between monocytes (CD14+ ) and MM cells (CD138+ ) influences the response to DARA. Further, the exposure of the BM samples to DARA is followed by the formation of a CD138+ CD14+ double-positive (DP) population, that quantitatively correlates with the anti-MM cells killing. These effects were dependent on the presence of a CD14+ CD16+ monocyte subset and on high CD16 expression levels. Lastly, the addition of a mAb neutralising the CD47/signal-regulatory protein α (SIRPα) axis was able to increase the killing mediated by DARA. The effects were observed only in coincidence with high CD14+ :CD138+ ratio, with a significant presence of the DP population and were correlated with CD16 expression. In conclusion, the present study underlines the critical role of the CD16+ monocytes in DARA anti-MM killing effects and gives a rationale to test the combination of an anti-CD47 mAb with anti-CD38 mAbs.

PD-L1/PD-1 Pattern of Expression Within the Bone Marrow Immune Microenvironment in Smoldering Myeloma and Active Multiple Myeloma Patients.

Background: The PD-1/PD-L1 axis has recently emerged as an immune checkpoint that controls antitumor immune responses also in hematological malignancies. However, the use of anti-PD-L1/PD-1 antibodies in multiple myeloma (MM) patients still remains debated, at least in part because of discordant literature data on PD-L1/PD-1 expression by MM cells and bone marrow (BM) microenvironment cells. The unmet need to identify patients which could benefit from this therapeutic approach prompts us to evaluate the BM expression profile of PD-L1/PD-1 axis across the different stages of the monoclonal gammopathies. Methods: The PD-L1/PD-1 axis was evaluated by flow cytometry in the BM samples of a total cohort of 141 patients with monoclonal gammopathies including 24 patients with Monoclonal Gammopathy of Undetermined Significance (MGUS), 38 patients with smoldering MM (SMM), and 79 patients with active MM, including either newly diagnosed or relapsed-refractory patients. Then, data were correlated with the main immunological and clinical features of the patients. Results: First, we did not find any significant difference between MM and SMM patients in terms of PD-L1/PD-1 expression, on both BM myeloid (CD14+) and lymphoid subsets. On the other hand, PD-L1 expression by CD138+ MM cells was higher in both SMM and MM as compared to MGUS patients. Second, the analysis on the total cohort of MM and SMM patients revealed that PD-L1 is expressed at higher level in CD14+CD16+ non-classical monocytes compared with classical CD14+CD16- cells, independently from the stage of disease. Moreover, PD-L1 expression on CD14+ cells was inversely correlated with BM serum levels of the anti-tumoral cytokine, IL-27. Interestingly, relapsed MM patients showed an inverted CD4+/CD8+ ratio along with high levels of pro-tumoral IL-6 and a positive correlation between %CD14+PD-L1+ and %CD8+PD-1+ cells as compared to both SMM and newly diagnosed MM patients suggesting a highly compromised immune-compartment with low amount of CD4+ effector cells. Conclusions: Our data indicate that SMM and active MM patients share a similar PD-L1/PD-1 BM immune profile, suggesting that SMM patients could be an interesting target for PD-L1/PD-1 inhibition therapy, in light of their less compromised and more responsive immune-compartment.

Novel targets for the treatment of relapsing multiple myeloma.

Introduction: Multiple myeloma (MM) is characterized by the high tendency to relapse and develop drug resistance. Areas covered: This review focused on the main novel targets identified to design drugs for the treatment of relapsing MM patients. CD38 and SLAMF7 are the main surface molecules leading to the development of monoclonal antibodies (mAbs) recently approved for the treatment of relapsing MM patients. B cell maturation antigen (BCMA) is a suitable target for antibody-drug conjugates, bispecific T cell engager mAbs and Chimeric Antigen Receptor (CAR)-T cells. Moreover, the programmed cell death protein 1 (PD)-1/PD-Ligand (PD-L1) expression profile by MM cells and their microenvironment and the use of immune checkpoints inhibitors in MM patients are reported. Finally, the role of histone deacetylase (HDAC), B cell lymphoma (BCL)-2 family proteins and the nuclear transport protein exportin 1 (XPO1) as novel targets are also underlined. The clinical results of the new inhibitors in relapsing MM patients are discussed. Expert opinion: CD38, SLAMF7, and BCMA are the main targets for different immunotherapeutic approaches. Selective inhibitors of HDAC6, BCL-2, and XPO1 are new promising compounds under clinical investigation in relapsing MM patients.

Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients.

C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138⁺ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14⁺CD16⁺ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.