INTRODUCTION:Anti-HER2 drugs are becoming an important therapeutic option for various solid tumors, increasing the need for HER2 status testing. Comprehensive genomic profiling (CGP) panels, including FoundationOne®CDx, are commonly used to assess ERBB2 (encoding for HER2) copy number alterations. We aimed to evaluate the analytical validity of FoundationOne®CDx assay, by comparing ERBB2 copy number data with traditional HER2 status by immunohistochemistry (IHC)/in situ hybridization (ISH) assays in a heterogeneous cohort of solid tumor samples. METHODS:We retrospectively reviewed the 531 cases evaluated by FoundationOne®CDx in our Institution, and HER2 status by IHC/ISH could be internally analyzed in 68 cases, including 31 (45.5%) gastroesophageal, 17 (25.0%) colorectal, four (5.8%) breast and two (2.9%) cholangiocarcinoma patients.Tumors with estimated ERBB2 copy number ⩾ 4 by FoundationOne®CDx, and tumors with strong and complete (3+) membranous staining by IHC and/or a HER2/CEP17 ratio ⩾2 by ISH were considered NGS positive and IHC/ISH positive, respectively. RESULTS:We identified 21 NGS positive cases (30.9%); IHC/ISH analysis confirming overexpression/amplification in 16 cases (sensitivity: 76.2%), while among the 47 NGS negative cases, 45 were confirmed by IHC/ISH results (specificity: 90%), with a positive predictive value of 76.2% and a negative predictive value of 95.7%. CONCLUSIONS:FoundationOne®CDx provides an accurate evaluation of ERBB2 copy number status and may represent a cost-effective option in metastatic cancer patients for whom NGS testing is recommended.

PURPOSE:In multiple myeloma, tumor cells reprogram metabolic pathways to sustain growth and monoclonal immunoglobulin production. This study examines acetyl-CoA carboxylase 1 (ACC1), the enzyme driving the rate-limiting step in de novo lipogenesis, in multiple myeloma metabolic reprogramming, particularly in c-MYC (MYC)-driven subtypes. EXPERIMENTAL DESIGN:ACC1 expression was evaluated across multiple myeloma genetic subgroups, focusing on MYC translocations. Functional studies using ACC1 inhibitors and genetic knockdown assessed multiple myeloma cell growth, lipid synthesis, and metabolic homeostasis in vitro and in vivo. The role of MYC overexpression in ACC1 sensitivity was examined, with palmitate rescue experiments. Lipidomic analysis and assessments of endoplasmic reticulum (ER) stress, protein translation, and oxidative damage elucidated underlying mechanisms. RESULTS:ACC1 was overexpressed in MYC-translocated multiple myeloma. Its inhibition or knockdown reduced multiple myeloma cell growth in vitro and in vivo, particularly in MYC-overexpressing cells. ACC1 knockdown suppressed de novo lipid synthesis, partially rescued by palmitate. Lipidomic disruptions increased cholesterol ester desaturation and altered phospholipid ratios, inducing ER stress, impaired translation, protein carbonylation, oxidative damage, and apoptosis. CONCLUSIONS:ACC1 is a metabolic vulnerability in MYC-driven multiple myeloma. Inhibiting ACC1 disrupts lipid homeostasis, induces ER stress, and causes oxidative damage, impairing cell survival. Targeting lipid synthesis pathways, especially in MYC-dependent subtypes, offers a promising therapeutic strategy for multiple myeloma.

587 Background: Collecting Duct Carcinoma (CDC) represents 1% of all renal cell carcinomas (RCC) and is characterized by aggressive clinical behavior and a particularly dismal prognosis. Cabozantinib and platinum-based chemotherapy are active therapeutic options, but survival remains poor with no novel agents approved for the treatment of metastatic disease. Antibody-Drug Conjugate (ADC) targeting Nectin-4 and TROP-2 are dramatically changing the therapeutic landscape of Urothelial Carcinoma (UC). Given the biological and clinical similarities between CDC and UC, we evaluated Nectin-4 and TROP-2 expression in CDC. Methods: CDC tissue samples were collected from patients enrolled in the CICERONE study (NCT05372302), a multicentric trial that aims to define the transcriptomic profile of CDC with the goal of changing the paradigm of CDC management by using biology-driven treatments. Nectin-4 and TROP-2 are surface proteins involved in cell adhesion and proliferation. Their expression was assessed by immunohistochemistry (IHC) using a validated assay with a Nectin-4 antibody (clone M22-321b41.1) and a TROP-2 antibody (Ab227689 by Abcam).Staining area was quantified as a percentage (0–100%) and considered positive if >10% of the tumor surface was stained. Staining intensity was dichotomized into positive and weakly positive, based on a clear distinction from the negative control. Results: 59 tissue samples from patients with diagnosis of CDC were considered eligible after centralized histological review. Fresh or archival tumor tissue was collected from either primary lesion or metastatic sites biopsies at baseline before the start of systemic therapy. Preliminary results demonstrated that Nectin-4 was expressed in 24 out of 59 samples (41%), while TROP-2 positivity was observed in 58/59 cases (98%). Conclusions: CDC appears to be a unique kidney tumor, which lies between UC and RCC clinically and biologically. Here we report for the first time the expression of two potential therapeutic targets in metastatic CDC, Nectin-4 and TROP-2, confirming the similarities between CDC and UC.Based on this encouraging data, along with the interesting results from EV-302 trial, we hypothesize that pembrolizumab in combination with Antibody-Drug Conjugate (ADC) could be active in mCDC and this combo will be assessed in the phase II RePRINT trial (NCT06302569). Clinical trial information: NCT05372302 .

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organelles and it has been found to be deregulated in multiple myeloma (MM) patients. Experimental evidence indicated that NEAT1 silencing negatively impacts proliferation and viability of MM cells, both in vitro and in vivo, suggesting a role in DNA damage repair (DDR). In order to elucidate the biological and molecular relevance of NEAT1 upregulation in MM disease we exploited the CRISPR/Cas9 synergistic activation mediator genome editing system to engineer the AMO-1 MM cell line and generate two clones that para-physiologically transactivate NEAT1 at different levels. NEAT1 overexpression is associated with oncogenic and prosurvival advantages in MM cells exposed to nutrient starvation or a hypoxic microenvironment, which are stressful conditions often associated with more aggressive disease phases. Furthermore, we highlighted the NEAT1 involvement in virtually all DDR processes through, at least, two different mechanisms. On one side NEAT1 positively regulates the posttranslational stabilization of essential PS proteins, which are involved in almost all DDR systems, thus increasing their availability within cells. On the other hand, NEAT1 plays a crucial role as a major regulator of a molecular axis that includes ATM and the catalytic subunit of DNA-PK kinase proteins, and their direct targets pRPA32 and pCHK2. Overall, we provided novel important insightsthe role of NEAT1 in supporting MM cells adaptation to stressful conditions by improving the maintenance of DNA integrity. Taken together, our results suggest that NEAT1, and probably PS organelles, could represent a potential therapeutic target for MM treatment.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström Macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. We first showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T (Treg) cells with respect to control mice. These findings were translated into the WM clinical setting, where the transcriptomic profiling of WM patients'-derived regulatory T cells (Tregs) unveiled a peculiar WM-devoted mRNA signature, with significant enrichment for NF-kB-mediated TNF-a signaling-, MAPK-, PI3K/AKT-related genes; paralleled by different Treg functional phenotype. We demonstrated a significantly higher Treg-induction, -expansion and -proliferation triggered by WM cells as compared to their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-to-T cell cross-talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis supporting WM cell-Treg cell interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

Supplementary Figures S1-S2 and Supplementary Tables S1-S5.

SIRT6 is a NAD + -consuming deacetylase with different abilities, including genomic stability and chromatin accessibility regulation. 1 Importantly, it plays a controversial role in cancer, acting as a tumor suppressor or oncogene depending on the tumors. 2 Such a pleiotropic effect gave rise to extensive efforts to understand the cellular mechanisms by which SIRT6 upsets each cancer speci fi cally. 3-11 We previously reported that multiple myeloma (MM) cells exhibit high levels of SIRT6 in response to their genomic instability. Indeed, SIRT6, by downregulating the expression of extracellular signal-regulated kinase signaling – related genes, suppresses ETS-domain transcription factor (ELK1) activity and increases DNA repair levels via Chk1, resulting in DNA-damaging agents ’ resistance. 12 Here, we investigated the role of SIRT6 in MM by focusing on its relationship with the splicing machinery. We initially

Recent studies have demonstrated the occurrence of somatic mutations of MYD88 and CXCR4 as key players in Waldenstrom's Macroglobulinemia (WM) pathogenesis and disease progression. Despite the significant improvement in the knowledge of the molecular mechanisms supporting WM biology whether immunosuppressive mechanisms could contribute to WM pathogenesis remains unexplored. We interrogated the transcriptome signatures of the bone marrow (BM) microenvironment of WM patients (n:22) as compared to healthy individuals (n:10), we found a significant enrichment for a Treg-signature, and for CD40/CD40L signaling-related genes (FDR <0.001; P <0.01). These findings were confirmed using a transgenic murine lymphoplasmacytic/WM model, where the BM immune microenvironment showed an increased number of infiltrating CD4+ T lymphocytes over CD8+ T cells. Further characterization of the T-cell compartment revealed the presence of abundant CD4+CD25+FoxP3+ Treg cells, with respect to control mice. Murine WM cells recruited a significantly higher number of more abundant Ki67+Treg cells, as compared to B lymphocytes from healthy mice. Collectively, these results suggest that immunosuppressive Treg cells may play in supporting WM disease biology. These findings prompted us to focus on Tregs and their role in supporting WM pathogenesis. We performed bulk RNA sequencing of Tregs isolated from both WM patients (n:14) and healthy donors/HD (n:8); showing a peculiar transcriptome signature characterizing WM-derived Tregs as compared to their normal cellular counterpart. WM patient-derived Tregs presented with transcriptome profiling enriched for FOXP3-target genes and to Treg induction, as compared to healthy donor-derived Tregs; with WM-Tregs showing a significant enrichment for genes related to interferon- and TNFα-related genes via NF-kB. The functional impact of Treg in WM biology was next studied. We observed a significantly higher ability of WM cells to favor both induction and expansion of CD4+CD25+FoxP3+ Tregs, as compared to HD-derived CD19+ B-cells; paralleled by a significantly higher increase of Ki67-positive Treg induced by WM cells as compared to HD-derived CD19+ B-cells. Same results were obtained using WM primary cells. Data were further corroborated by demonstrating significant enrichment of several pro-proliferative- and pro-survival pathways in WM-Tregs, versus HD-counterpart, including MAPK- and PI3K/AKT-related genes. To gain insight into the potential molecular mechanisms responsible for the observed Treg induction in WM, we performed scRNAseq using the whole cell population harvested at the end of the induction assay. We evaluated the B-T-cell interactions at single cell level, adopting a B->T cross-talk model; and identified a set of four high-priority genes (CD40, TNF, TNFSF14, ICAM2): although all of them were expressed in B cells, only the expression of CD40 was maximal and virtually restricted to the B clusters, with the other three genes showing a more diffuse pattern. Importantly, CD40-ligand was shown to be the interactor expressed within the Treg cell compartment. Given the identified CD40/CD40L axis as a potential regulator of the WM cell/Treg cross-talk, we used the CD40/CD40L inhibitor DRI-C21045; and found a significant decrease in Treg induction and Treg proliferation. Halting CD40/CD40L interaction inhibited Treg induction and growth, also within the context of CXCR4-mutated WM, supported by inhibition of p-AKT and p-ERK in Treg cells. CD40/CD40L blockade led to inhibition of WM cell growth. Overall our studies have demonstrated the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

PURPOSE:The NONO protein belongs to the multifunctional family of proteins that can bind DNA, RNA and proteins. It is located in the nucleus of most mammalian cells and can affect almost every step of gene regulation. Dysregulation of NONO has been found in many types of cancer; however, data regarding its expression and relevance in Multiple Myeloma (MM) are virtually absent.METHODS:We took advantage of a large cohort of MM patients enrolled in the Multiple Myeloma Research Foundation CoMMpass study to elucidate better the clinical and biological relevance of NONO expression in the context of the MM genomic landscape and transcriptome.RESULTS:NONO is overexpressed in pathological samples compared to normal controls. In addition, higher NONO expression levels are significant independent prognostic markers of worse clinical outcome in MM. Our results indicate that NONO deregulation may play a pathogenetic role in MM by affecting cell cycle, DNA repair mechanisms, and influencing translation by regulating ribosome biogenesis and assembly. Furthermore, our data suggest NONO involvement in the metabolic reprogramming of glucose metabolism from respiration to aerobic glycolysis, a phenomenon known as the 'Warburg Effect' that supports rapid cancer cell growth, survival, and invasion.CONCLUSION:These findings strongly support the need of future investigations for the understanding of the mechanisms of deregulation and the biological role and activity of NONO in MM.

Abstract Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3′-UTR region of the IL-12Rβ1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rβ1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rβ1–positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Significant skeletal alterations are present in Chronic Lymphocytic Leukemia (CLL) patients; bone erosion, particularly evident in the long bone shaft, appeared increased in the progressive disease stage. Moreover, the partial colonization of the bone with reactive bone marrow we documented via PET-FDG imaging suggests that neoplastic cell overgrowth contributes to bone derangement. Indeed, cytokines released by leukemic B cells impair osteoblast differentiation and enhance osteoclast formation in vitro. CD16, Fcγ-RIIIa, has been previously indicated as a marker of osteoclast precursors. We demonstrate, here, that the percentage of circulating monocytes, CD16+, is significantly higher in CLL patients than in normal controls and directly correlated with the extent of bone erosion. When we assessed if healthy monocytes, treated with a CLL-conditioned medium, modulated RANK, RANKL and CD16, we observed that all these molecules were up-regulated and CD16 to a greater extent. Altogether, these findings suggest that leukemic cells facilitate osteoclast differentiation. Interestingly, the evidence that monocytes, polarized toward the M2 phenotype, were characterized by high CD16 expression and showed a striking propensity to differentiate toward osteoclasts may provide further explanations for the enhanced levels of bone erosion detected, in agreement with the high number of immunosuppressive-M2 cells present in these patients.

e16507 Background: The phase II BONSAI trial ( n = 25 ; NCT 03354884) met the primary endpoint demonstrating activity of cabozantinib in untreated metastatic collecting ducts carcinoma (mCDC), a rare and biologically poorly characterized disease. Here we report on molecular analyses of baseline tissue samples. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 18 mCDC patients enrolled in BONSAI were sequenced by TruSeq RNA Exome kit (Illumina). The data were mapped and quantified using STAR and htseq, respectively. Globaltest and edgeR packages in R were used to assess the correlation between transcriptional profiles and survival data. Nineteen samples underwent DNA Sequencing with the Oncomine Comprehensive Assay Plus (ThermoFisher Scientific). The reads were aligned to the human genome reference (hg19) and analyzed with Opencravat and IonReporter software. Germline variants were excluded based on 1000 Genomes, GnomAd and Exac databases, and Clinical annotation of somatic variants was performed using ClinVar. Results: The global expression levels of thirty-one genes have been found significantly associated with overall survival (OS). The natural grouping of the 18 tumor samples based on the 31-gene signature identified a main group of 11 cases, showing global higher expression levels in 22 out of the 31 genes. This group displayed overall a significant higher OS rate in comparison to the remaining 7 patients, carrying opposite expression trend and mostly undergoing to disease progression. The identified signature was enriched in biological processes like cell junction, cytoskeleton organization and methylation. FOS oncogene was among the 9 genes negatively associated to OS, showing common higher expression in the poorer survival rate group. Furthermore, a 22-gene signature was found significantly associated with progression-free survival (PFS), involving mainly genes associated to cell cycle regulation or recognized as components of Golgi apparatus. Only three genes were globally up-regulated in the group of 9 patients characterized by shorter PFS. Finally, heterogeneous pattern of somatic mutations was identified in 19 tumor samples, with at least 8 genes recurrently affected in more than three patients. Notably, mutated genes were mostly involved in DNA repair and chromatin modification processes. Conclusions: Our findings for the first time define specific molecular signatures that differentiate therapy-specific outcomes in first-line mCDC, warranting further investigation of their involvement in the tumor biology. Clinical trial information: NCT 03354884.

•Limited data are available about the prognostic impact of CALR mutations subtypes in essential thrombocythemia (ET). •We studied the impact of CALR mutations in a monocentric cohort of CALR-mutated ET. •A lower TFS, albeit with borderline significance, was found in the type 1 CALR group compared with both type 2 and "other". •Our data suggest how specific CALR mutations may impact on thrombotic risk in ET.

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for “priming” MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.

The mitochondrial quality control network includes several epigenetically-regulated genes involved in mitochondrial dynamics, mitophagy, and mitochondrial biogenesis under physiologic conditions. Dysregulated expression of such genes has been reported in various disease contexts, including cancer. However, their expression pattern and the possible underlying epigenetic modifications remain to be defined within plasma cell (PC) dyscrasias. Herein, we compared the mRNA expression of mitochondrial quality control genes from multiple myeloma, plasma cell leukemia patients and human myeloma cell lines (HMCLs) with healthy plasma cells; moreover, by applying the Sequenom MassARRAY EpiTYPER technology, we performed a pilot investigation of their CpG methylation status in HMCLs. Overall, the results provided indicate dysregulated expression of several mitochondrial network’s genes, and alteration of the CpG methylation profile, underscoring novel potential myeloma biomarkers deserving in-depth functional investigation in the future.

Despite recent therapeutic advances, multiple myeloma (MM) patients experience relapses as they become resistant to various classes and combinations of treatment. Melphalan (L-PAM) is an ageless drug. However, its use in the autologous stem cell transplantation (ASCT) setting and the innovative quadruplet regimen as well as daratumumab, bortezomib, and prednisone make this old drug current yet. Melflufen is a peptide-conjugated alkylator belonging to a novel class of compounds, representing an overcoming of L-PAM in terms of mechanism of action and effectiveness. The improved melflufen cytotoxicity is related to aminopeptidase activity, notably present in normal and neoplastic cells and remarkably heavily overexpressed in MM cells. Upon entering a cell, melflufen is cleaved by aminopeptidases, ultimately releasing the L-PAM payload and eliciting further the inflow and cleavage of the conjugated peptide. This virtuous loop persists until all extracellular melflufen has been utilized. The aminopeptidase-driven accumulation results in a 50-fold increase in L-PAM cell enrichment as compared with free alkylator. This condition produces selective cytotoxicity, increased on-target cell potency, and decreased off-target cell toxicity, ultimately overcoming resistance pathways triggered by previous treatments, including alkylators. Due to its distinct mechanism of action, melflufen plus dexamethasone as a doublet, and in combination with other novel drugs, has the potential to be beneficial for a broad range of patients with relapsed/refractory (RR) MM in third- or even in second-line therapy. The safety profile of melflufen has been consistent across studies, and no new safety concerns have been identified when melflufen was administered in doublet and triplet combinations. Based on growing clinical evidence, melflufen could be not only a good addition in the fight against RRMM but also a drug with a very favorable tolerability profile.