Identification of the Cysteine Protease Legumain as a Potential Chronic Hypoxia-Specific Multiple Myeloma Target Gene.

Multiple myeloma (MM) is the second most common hematologic malignancy, which is characterized by clonal proliferation of neoplastic plasma cells in the bone marrow. This microenvironment is characterized by low oxygen levels (1-6% O2), known as hypoxia. For MM cells, hypoxia is a physiologic feature that has been described to promote an aggressive phenotype and to confer drug resistance. However, studies on hypoxia are scarce and show little conformity. Here, we analyzed the mRNA expression of previously determined hypoxia markers to define the temporal adaptation of MM cells to chronic hypoxia. Subsequent analyses of the global proteome in MM cells and the stromal cell line HS-5 revealed hypoxia-dependent regulation of proteins, which directly or indirectly upregulate glycolysis. In addition, chronic hypoxia led to MM-specific regulation of nine distinct proteins. One of these proteins is the cysteine protease legumain (LGMN), the depletion of which led to a significant growth disadvantage of MM cell lines that is enhanced under hypoxia. Thus, herein, we report a methodologic strategy to examine MM cells under physiologic hypoxic conditions in vitro and to decipher and study previously masked hypoxia-specific therapeutic targets such as the cysteine protease LGMN.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells.

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 µM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

Characteristic bone marrow findings in patients with UBA1 somatic mutations and VEXAS syndrome.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a newly characterized syndrome with underlying somatic UBA1 mutations in myeloid cells linking hematologic disease with autoinflammatory rheumatologic disorders. Hematologic abnormalities, particularly peripheral blood cytopenia(s) may prompt bone marrow evaluation in patients with known or unrecognized VEXAS syndrome. This review highlights key findings and diagnostic considerations encountered during bone marrow examination in patients with this disorder. Frequently reported hematologic changes include macrocytic anemia, cytoplasmic vacuoles in myeloid and erythroid precursors, marrow hypercellularity, and varying degrees of dysplasia. Myelodysplastic syndrome and plasma cell neoplasms have been diagnosed in patients with VEXAS syndrome. Macrophage activation syndrome and/or hemophagocytic lymphohistiocytosis and monoclonal B-cell lymphocytosis have also been reported. The bone marrow is a target organ in VEXAS syndrome. Heightened awareness of the bone marrow features and hematologic complications may aid in identifying individuals with VEXAS who may benefit from increased disease surveillance or alternative therapeutic strategies.

Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma.

Multiple myeloma (MM) is a heterogeneous disease characterized by significant genomic instability. Recently, a causal role for the AID/APOBEC deaminases in inducing somatic mutations in myeloma has been reported. We have identified APOBEC/AID as a prominent mutational signature at diagnosis with further increase at relapse in MM. In this study, we identified upregulation of several members of APOBEC3 family (A3A, A3B, A3C, and A3G) with A3G, as one of the most expressed APOBECs. We investigated the role of APOBEC3G in MM and observed that A3G expression and APOBEC deaminase activity is elevated in myeloma cell lines and patient samples. Loss-of and gain-of function studies demonstrated that APOBEC3G significantly contributes to increase in DNA damage (abasic sites and DNA breaks) in MM cells. Evaluation of the impact on genome stability, using SNP arrays and whole genome sequencing, indicated that elevated APOBEC3G contributes to ongoing acquisition of both the copy number and mutational changes in MM cells over time. Elevated APOBEC3G also contributed to increased homologous recombination activity, a mechanism that can utilize increased DNA breaks to mediate genomic rearrangements in cancer cells. These data identify APOBEC3G as a novel gene impacting genomic evolution and underlying mechanisms in MM.

PRMT5 regulates cell pyroptosis by silencing CASP1 in multiple myeloma.

Protein arginine methyltransferase 5 (PRMT5), a histone methyltransferase responsible for the symmetric dimethylation of histone H4 on Arg 3 (H4R3me2s), is an enzyme that participates in tumor cell progression in a variety of hematological malignancies. However, the biological functions of PRMT5 in multiple myeloma (MM) and the underlying molecular mechanisms remain unclear. In this study, we conducted a bioinformatics analysis and found that PRMT5 expression was significantly upregulated in MM. In vitro and in vivo phenotypic experiments revealed that knockdown of PRMT5 expression enhanced cell pyroptosis in MM. Moreover, we found that CASP1 expression was negatively correlated with PRMT5 expression, and repressing PRMT5 expression rescued both the phenotype and expression markers (N-GSDMD, IL-1b, and IL-18). Inhibition of PRMT5 activity increased CASP1 expression and promoted MM cell pyroptosis. Finally, high expression of PRMT5 or low expression of CASP1 was correlated with poor overall survival in MM. Collectively, our results provide a mechanism by which PRMT5 regulates cell pyroptosis by silencing CASP1 in MM.

Caspase-8 Regulates the Antimyeloma Activity of Bortezomib and Lenalidomide.

Proteasome inhibitors and immunomodulatory drugs (IMiDs) are two major types of drugs for the treatment of multiple myeloma. Although different combination therapies for myeloma have been developed and achieved high responsive rate, these strategies frequently result in drug resistance. Therefore, it is necessary to explore new molecular mechanisms and therapeutic approaches to fulfill this unmet medical need. Here, we find that proteasome inhibitor bortezomib (Btz) causes cereblon (CRBN) cleavage and that caspase-8 (CASP-8) is responsible for this cleavage. Either inhibition or genetic depletion of CASP-8 decreased the CRBN cleavage upon Btz treatment, which could potentiate the antimyeloma activity of IMiD lenalidomide (Len). This work suggests that administration of CASP-8 inhibitors might enhance the overall effectiveness of Btz/Len-based therapeutic treatment of patients with myeloma. SIGNIFICANCE STATEMENT: Caspase-8 activation upon bortezomib treatment results in the cleavage of cereblon, a substrate receptor of the cullin-4 RING E3 ligase, which is responsible for the degradation of two transcription factors, Ikaros family zinc finger protein (IKZF) 1 and IKZF3, in the presence of immunomodulatory drugs including lenalidomide. The administration of caspase-8 inhibitor may enhance the antimyeloma activity of the combination therapy with bortezomib and lenalidomide.

Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma.

Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and ß-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.

Phosphatase PP2A enhances MCL-1 protein half-life in multiple myeloma cells.

Multiple myeloma (MM), a treatable but incurable malignancy, is characterized by the growth of clonal plasma cells in protective niches in the bone marrow. MM cells depend on expression of BCL-2 family proteins, in particular MCL-1, for survival. The regulation of MCL-1 is complex and cell type-dependent. Unraveling the exact mechanism by which MCL-1 is overexpressed in MM may provide new therapeutic strategies for inhibition in malignant cells, preferably limiting side effects in healthy cells. In this study, we reveal that one cause of overexpression could be stabilization of the MCL-1 protein. We demonstrate this in a subset of MM and diffuse large B cell lymphoma (DLBCL) cell lines and MM patient samples. We applied a phosphatase siRNA screen to identify phosphatases responsible for MCL-1 stabilization in MM, and revealed PP2A as the MCL-1 stabilizing phosphatase. Using the PP2A inhibitor okadaic acid, we validated that PP2A dephosphorylates MCL-1 at Ser159 and/or Thr163, and thereby stabilizes MCL-1 in MM cells with long MCL-1 half-life, but not in DLBCL cells. Combined kinase and phosphatase inhibition experiments suggest that the MCL-1 half-life in MM is regulated by the counteracting functions of JNK and PP2A. These findings increase the understanding of the mechanisms by which MCL-1 is post-translationally regulated, which may provide novel strategies to inhibit MCL-1 in MM cells.

Inhibition of the ʟ-glutamine transporter ASCT2 sensitizes plasma cell myeloma cells to proteasome inhibitors.

Proteasome inhibitors (PIs), used in the treatment of plasma cell myeloma (PCM), interfere with the degradation of misfolded proteins leading to activation of unfolded protein response (UPR) and cell death. However, despite initial strong antimyeloma effects, PCM cells eventually develop acquired resistance to PIs. The pleiotropic role of ʟ-glutamine (Gln) in cellular functions makes inhibition of Gln metabolism a potentially good candidate for combination therapy. Here, we show that PCM cells, both sensitive and resistant to PIs, express membrane Gln transporter (ASCT2), require extracellular Gln for survival, and are sensitive to ASCT2 inhibitors (ASCT2i). ASCT2i synergistically potentiate the cytotoxic activity of PIs by inducing apoptosis and modulating autophagy. Combination of ASCT2 inhibitor V9302 and proteasome inhibitor carfilzomib upregulates the intracellular levels of ROS and oxidative stress markers and triggers catastrophic UPR as shown by upregulated spliced Xbp1 mRNA, ATF3 and CHOP levels. Moreover, analysis of RNA sequencing revealed that the PI in combination with ASCT2i reduced the levels of Gln metabolism regulators such as MYC and NRAS. Analysis of PCM patients' data revealed that upregulated ASCT2 and other Gln metabolism regulators are associated with advanced disease stage and with PIs resistance. Altogether, we identified a potent therapeutic approach that may prevent acquired resistance to PIs and may contribute to the improvement of treatment of patients suffering from PCM.

Suppression of steroid 5α-reductase type I promotes cellular apoptosis and autophagy via PI3K/Akt/mTOR pathway in multiple myeloma.

Steroid 5α-reductase type I (SRD5A1) is a validated oncogene in many sex hormone-related cancers, but its role in multiple myeloma (MM) remains unknown. Based on gene expression profiling (GEP) of sequential MM samples during the disease course, we found that the aberrant expression of SRD5A1 was correlated with progression and poor prognosis in MM patients. In this study, the oncogenic roles of SRD5A1 were validated in human MM cell lines (ARP1 and H929) and the xenograft MM model as well as the 5TMM mouse model. MTT and flow cytometry were used to assess MM cell proliferation, cell cycle, and apoptosis post inducible knockdown SRD5A1 by lentivirus-mediated short-hairpin RNA (shRNA). Transcriptomic sequencing, immunofluorescence, and western blot were used to investigate the effects of SRD5A1 suppression on cell apoptosis and autophagy. Mechanistically, SRD5A1 downregulation simultaneously regulated both the Bcl-2 family protein-mediated apoptosis and the autophagic process via PI3K/Akt/mTOR signaling pathway in MM cells. Meanwhile, the autophagy inhibitor (3-methyladenine) and SRD5A1 inhibitor (Dutasteride) were utilized to evaluate their anti-myeloma effect. Thus, our results demonstrated that SRD5A1 downregulation simultaneously regulated both the apoptosis and the autophagic process in MM cells. The dual autophagy-apoptosis regulatory SRD5A1 may serve as a biomarker and potential target for MM progression and prognosis.

A phase I, dose-escalation study of oral PIM447 in Japanese patients with relapsed and/or refractory multiple myeloma.

PIM447, a pan-proviral integration site for Moloney leukemia (PIM) kinase inhibitor, has shown preclinical activity in multiple myeloma (MM). This phase I, open-label, multicenter, dose-escalation study aimed to determine the maximum tolerated dose (MTD) and recommended dose for expansion (RDE) of PIM447 in Japanese patients with relapsed and/or refractory (R/R) MM. The study included 13 patients (250 mg once daily (QD), [n = 7]; 300 mg QD, [n = 6]). The sole dose-limiting toxicity observed was grade 3 QTc prolongation in one patient from the 300 mg group, and the MTD and RDE was not determined. The most common suspected PIM447-related adverse events (AEs) included thrombocytopenia (76.9%), anemia (53.8%), and leukopenia (53.8%). All patients experienced at least one grade 3 or 4 AE, most frequently thrombocytopenia or leukopenia (61.5% each). The overall response rate was 15.4%, disease control rate 69.2%, clinical benefit rate 23.1%, and two patients had a partial response (one in each dose group). Two patients treated with 250 mg QD had a progression-free survival > 6 months. PIM447 250 mg or 300 mg QD was tolerated in Japanese patients with R/R MM. Further studies are required to evaluate clinical outcomes of PIM447 in combination with other drugs for the treatment of MM.Trial registration: clinicaltrials.gov: (NCT02160951).

Updates to the drug-resistant mechanism of proteasome inhibitors in multiple myeloma.

Proteasome inhibitors (PIs) have been a kind of backbone therapies for newly diagnosed as well as relapsed or refractory myeloma patients in the last two decades. Bortezomib, the first-in-class PI, was approved by the United States Food and Drug Administration in 2003. The key roles of this class of agents are targeting at the overstressed 26S proteasome, which are involved in the pathogenesis of the disease. Despite recent advancements in clinical antimyeloma treatment, the acquisition of resistance is a major limitation in PI therapy. This review aims at a better understanding of the pathways and biomarkers involved in MM drug resistance.

Protein tyrosine phosphatases in multiple myeloma.

Many cell signaling pathways are activated or deactivated by protein tyrosine phosphorylation and dephosphorylation, catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. Even though PTPs are as important as PTKs in this process, their role has been neglected for a long time. Multiple myeloma (MM) is a cancer of plasma cells, which is characterized by production of monoclonal immunoglobulin, anemia and destruction of bone. MM is still incurable with high relapse frequency after treatment. In this review, we highlight the PTPs that were previously described in MM or have a role that can be relevant in a myeloma context. Our purpose is to show that despite the importance of PTPs in MM pathogenesis, many unanswered questions in this field need to be addressed. This might help to detect novel treatment strategies for MM patients.

A new parameter in multiple myeloma: CYP3A4*1B single nucleotide polymorphism.

Multiple myeloma (MM) is a disease caused by malignant plasma cells, causing free light chain release accompanying the increase in monoclonal immunoglobulin. Cytochrome P450 (CYP) is one of the large and functional enzyme families composed of various hemoproteins. This protein network has been shown to play a role in many treatment steps in current practices. We aimed to investigate the relationship between genotypes of CYP3A4*1B and treatment response and prognosis of MM. Seventy-two patients diagnosed with MM between January 2016 and 2020 and 100 healthy people to create a control group participated in our study. Genotypes were classified in 3 separate groups as NN, MN, and MM. Both PFS and OS were significantly higher in the NN genotype (p = 0.001, p = 0.014). Being under the age of 65 was 27.988 times more protective for OS and 4.496 times for PFS (p = 0.006, p = 0.017). NN genotype was shown to be 41.666-fold protective for OS and 3.144-fold protective for PFS (p = 0.004, p = 0.030). This study demonstrated that CYP3A4*1B NN genotype, which is an important cytochrome p450 member for the treatment of MM, was 41.666-fold protective for OS and 3.144-fold protective for PFS. It was shown in this study for the first time in the literature as a valuable contribution.

GSK3α: An Important Paralog in Neurodegenerative Disorders and Cancer.

The biological activity of the enzyme glycogen synthase kinase-3 (GSK3) is fulfilled by two paralogs named GSK3α and GSK3ß, which possess both redundancy and specific functions. The upregulated activity of these proteins is linked to the development of disorders such as neurodegenerative disorders (ND) and cancer. Although various chemical inhibitors of these enzymes restore the brain functions in models of ND such as Alzheimer's disease (AD), and reduce the proliferation and survival of cancer cells, the particular contribution of each paralog to these effects remains unclear as these molecules downregulate the activity of both paralogs with a similar efficacy. Moreover, given that GSK3 paralogs phosphorylate more than 100 substrates, the simultaneous inhibition of both enzymes has detrimental effects during long-term inhibition. Although the GSK3ß kinase function has usually been taken as the global GSK3 activity, in the last few years, a growing interest in the study of GSK3α has emerged because several studies have recognized it as the main GSK3 paralog involved in a variety of diseases. This review summarizes the current biological evidence on the role of GSK3α in AD and various types of cancer. We also provide a discussion on some strategies that may lead to the design of the paralog-specific inhibition of GSK3α.

Downregulation of PA28α induces proteasome remodeling and results in resistance to proteasome inhibitors in multiple myeloma.

Protein homeostasis is critical for maintaining eukaryotic cell function as well as responses to intrinsic and extrinsic stress. The proteasome is a major portion of the proteolytic machinery in mammalian cells and plays an important role in protein homeostasis. Multiple myeloma (MM) is a plasma cell malignancy with high production of immunoglobulins and is especially sensitive to treatments that impact protein catabolism. Therapeutic agents such as proteasome inhibitors have demonstrated significant benefit for myeloma patients in all treatment phases. Here, we demonstrate that the 11S proteasome activator PA28α is upregulated in MM cells and is key for myeloma cell growth and proliferation. PA28α also regulates MM cell sensitivity to proteasome inhibitors. Downregulation of PA28α inhibits both proteasomal load and activity, resulting in a change in protein homeostasis less dependent on the proteasome and leads to cell resistance to proteasome inhibitors. Thus, our findings suggest an important role of PA28α in MM biology, and also provides a new approach for targeting the ubiquitin-proteasome system and ultimately sensitivity to proteasome inhibitors.

In vitro and in vivo efficacy of the novel oral proteasome inhibitor NNU546 in multiple myeloma.

Proteasome inhibition demonstrates highly effective impact on multiple myeloma (MM) treatment. Here, we aimed to examine anti-tumor efficiency and underlying mechanisms of a novel well tolerated orally applicable proteasome inhibitor NNU546 and its hydrolyzed pharmacologically active form NNU219. NNU219 showed more selective inhibition to proteasome catalytic subunits and less off-target effect than bortezomib ex vivo. Moreover, intravenous and oral administration of either NNU219 or NNU546 led to more sustained pharmacodynamic inhibitions of proteasome activities compared with bortezomib. Importantly, NNU219 exhibited potential anti-MM activity in both MM cell lines and primary samples in vitro. The anti-MM activity of NNU219 was associated with induction of G2/M-phase arrest and apoptosis via activation of the caspase cascade and endoplasmic reticulum stress response. Significant growth-inhibitory effects of NNU219 and NNU546 were observed in 3 different human MM xenograft mouse models. Furthermore, such observation was even found in the presence of a bone marrow microenvironment. Taken together, these findings provided the basis for clinical trial of NNU546 to determine its potential as a candidate for MM treatment.

Blockade of PLD1 potentiates the antitumor effects of bortezomib in multiple myeloma cells by inhibiting the mTOR/NF-κB signal pathway.

OBJECTIVE: Phospholipase D1 (PLD1) is an enzyme of the phospholipase D (PLD) superfamily. It is involved in the occurrence of various tumors. However, its role in multiple myeloma (MM) remained undefined. This study aimed to investigate the mechanism of PLD1 in the therapy of myeloma disease. MATERIAL AND METHODS: Cell lines U266 and H929 were treated with PLD1 specific inhibitor VU0359595 combined bortezomib, a proteasome inhibitor. Their effects on MM cell proliferation, apoptosis, and relevant signal pathways of apoptosis were determined by cell counting kit-8 (CCK-8), real-time polymerase reaction chain (RT-PCR), ATP assay, and western blot. RESULTS: PLD1 was highly expressed in U266 and H929 cells. VU0359595 didn't affect the proliferation and apoptosis of MM cells. However, VU0359595 could enhance growth inhibition, decreasing mitochondrial membrane potentials (MMPs) and ATP levels of bortezomib treated MM cells. VU0359595 also strengthened bortezomib-induced apoptosis via activating caspase-8, caspase-9, caspase-3; and down-regulating the expressions of anti-apoptosis proteins BCL-2. In addition, the bortezomib-induced cytotoxicity on MM cells was significantly augmented by VU0359595 through efficient suppression of the mTOR/NF-κB signal pathway. CONCLUSION: PLD1 inhibition can remarkably exert antitumor effects with bortezomib on MM, which is a novel potentially targeting therapeutic agent, especially for drug-resistant MM patients.

Targeting of the E3 ubiquitin-protein ligase HUWE1 impairs DNA repair capacity and tumor growth in preclinical multiple myeloma models.

Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase ß, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.