Multiple myeloma (MM), characterised by the clonal proliferation of plasma cells in the bone marrow, is the second most common haematological malignancy worldwide. Although there is now an impressive artillery of therapeutics to tackle this condition, resistance remains a prevalent issue. The bone marrow microenvironment performs a crucial role in supporting MM pathogenesis and promoting the development of therapeutic resistance. Extracellular vesicles (EVs), small vesicles that carry bioactive molecules, are a key component of cell-to-cell communication within the bone marrow microenvironment. In this review, we summarise the contribution of EVs to disease progression and anticancer treatment resistance and discuss the potential therapeutic applications of EVs in MM.

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.

Introduction Whilst rare, the transformation to accelerated or blast phase of a myeloproliferative neoplasm (AP/BP-MPN) is devastating. Patients rarely present with chemo-sensitive disease, and median survival rates in those treated with intensive or non-intensive regimens are limited to only a few months. New tolerable treatment approaches that target and eradicate the mutant clone are therefore urgently required. Interferon alpha (IFN-α) is a frontline therapy in chronic phase MPN and can induce sustained molecular responses in JAK2 V617F positive patients through targeted effects on the mutant clonal population. Unfortunately, these responses are modest in most cases and are gradual, taking many years to develop. Identification of new combination treatments which potentiate the effect of IFN-α in MPN cells may offer a new disease modifying approach to benefit patients with advanced and aggressive MPN phenotypes. Aim IFN-α was observed to induce apoptosis in JAK2 V617F positive cell line models. We set out to establish the mechanism of apoptosis induced in these cells to identify and exploit IFN-α induced therapeutic vulnerabilities with rational combination treatments. Results We developed an IFN-α resistant UKE1 MPN cell line (UKE1-NR) and directly compared it to the IFN-α responsive parental model (UKE1-P) by RNA-Seq. Following exogenous IFN-α therapy, both UKE1-P and UKE1-NR upregulated canonical IFN pathways and increased phosphorylation of STAT1 to a similar extent. Responsive UKE1-P cells demonstrated transcriptional upregulation of the PERK/ATF4/CHOP arm of the unfolded protein response (UPR) and the pro-apoptotic gene PMAIP1 (NOXA), which has previously been identified as a downstream target of this pathway. This was not observed in the UKE1-NR cells. Using qPCR, we validated this in JAK2 V617F positive UKE1, HEL and SET2 cells, observing upregulation of the UPR genes ATF3 and DDIT3 (CHOP) as well as PMAIP1 (NOXA) following IFN-α exposure. Using ATAC-seq, we observed loss of chromatin accessibility in the UKE1-NR cells at genetic loci identified as binding sites for ATF3, ATF4 and CHOP, consistent with a functional role for these transcription factors. Small molecule inhibition of ATF4 activation using ISRIB antagonised the effect of IFN-α in responsive UKE1-P cells highlighting an important role for this pathway in mediating the response. Bortezomib (Bz), a proteasome inhibitor, is a known inducer of the UPR. Combination of Bz with IFN-α synergistically increased apoptosis in all three JAK2 V617F cell line models. Furthermore, combination therapy resulted in significantly increased expression of ATF3, DDIT3 (CHOP) and PMAIP1 (NOXA) in comparison to single agent treatment. Importantly this combination was also determined to be synergistic in UKE1-NR cells despite non-response to single agent IFN-α. Moreover, the efficacy of this combination appears to be p53-independent as it was observed in TP53 wild type (UKE1) and mutant (HEL/SET2) cells. Additionally, the combination is effective following CRISPR knockdown of TP53 in HEL cells. CRISPR knockout of PMAIP1 (NOXA) subsequently demonstrated partial dependence on NOXA for the effectiveness of this combination. NOXA upregulation can sensitise to BCL2 inhibition, and in keeping with this, we have observed enhanced sensitivity to the clinically-relevant BCL2 inhibitor venetoclax and synergy in combination with Bz/IFN-α. Conclusion IFN-α can induce apoptosis in a range of JAK2 V617F positive cells. We have demonstrated that this apoptosis is characterised by a transcriptional upregulation of the UPR and NOXA and induces a therapeutic vulnerability which can be effectively exploited in combination therapy with bortezomib in a TP53 independent manner. These early pre-clinical results using readily available drug combinations offer a potential novel therapeutic approach in combination with or without BCL2 inhibition for patients with aggressive forms of MPN including AP/BP-MPN and will be taken forward for further study.

Despite significant improvements in treatment strategies over the past couple of decades, multiple myeloma (MM) remains an incurable disease due to the development of drug resistance. Metabolic reprogramming is a key feature of cancer cells, including MM, and acts to fuel increased proliferation, create a permissive tumour microenvironment, and promote drug resistance. This review presents an overview of the key metabolic adaptations that occur in MM pathogenesis and in the development of resistance to proteasome inhibitors, the backbone of current MM therapy, and considers the potential for therapeutic targeting of key metabolic pathways to improve outcomes.

Commentary on: Xiong Y, Wang L, Xu S, Fu B, Che Y, Zaky MY, Tian R, Yao R, Guo D, Sha Z, Lin F, Lin X, Wu H. Small molecule Z363 co-regulates TAF10 and MYC via the E3 ligase TRIP12 to suppress tumour growth. Clin Transl Med. 2023;13(1):e1153. doi: 10.1002/ctm2.1153 MYC is a pleiotropic transcription factor involved in the regulation of critical cellular processes including cell proliferation, differentiation and apoptosis. Expression and activity of MYC are deregulated in over 70% of human malignancies, contributing to both tumour initiation and maintenance, and are frequently associated with poor prognosis. This central role in oncogenesis makes MYC a highly desirable therapeutic target. Direct targeting of MYC on a protein level is challenging due to the disordered nature of the protein structure and lack of enzymatic activity, nonetheless, the first direct inhibitor Omomyc, a dominant-negative mutant of MYC, recently entered clinical trials.1 In addition, numerous alternative strategies to target MYC transcription, translation, stability and interaction with dimerization partner MAX are also under investigation. One promising approach to indirectly target MYC is to pursue molecules responsible for its post-translational regulation (Figure 1). To date, at least 18 E3 ligases have been identified to mediate MYC ubiquitination, functioning to either regulate MYC stability or activity and are considered potentially exploitable targets. For example, the natural diterpenoid compound oridonin activates the E3 ligase SCFFBW7 leading to targeted degradation of MYC through the proteasome.2 On the other hand, inhibition of the E3 ligase HUWE1, which acts to promote MYC function, results in suppression of MYC-dependent transactivation, blocks the formation of protective MYC multimers at the replication fork and enhances MYC degradation.3-5 Another emerging strategy to promote MYC degradation is using proteolysis targeting chimaeras (PROTACs). These are heterobifunctional small molecules that simultaneously bind a target and an E3 ligase to induce the ubiquitination and degradation of the target protein. So far, no PROTACS have been developed to induce direct degradation of MYC, but PROTACS targeting bromodomain and extra-terminal domain (BET) proteins indirectly reduce MYC expression and show promising pre-clinical activity.6, 7 A recent study from Xiong et al. identified a small molecule, Z363, that promotes the degradation of MYC and TAF10 via the E3 ligase TRIP12. The authors demonstrated elevated expression of MYC and the transcription factor TAF10 across a range of primary tumour types when compared to adjacent non-tumour tissues and hypothesised that TAF10 could enhance the transcriptional activity of MYC. Accordingly, knockdown and overexpression studies led to decreased MYC expression and increased MYC promoter activity, respectively. Mutation studies in the breast cancer cell line MCF7 identified that the MBIV domain on MYC was essential for this interaction. The MBIV domain has been reported to enhance the transcription of 150 genes, however, the effects of deletion of this domain were variable across model systems suggesting that it may only be required for transformation in specific biological contexts.8 Further understanding of the functional significance of this domain across breast cancer and other cancer types would be of great interest. Xiong and colleagues next screened a panel of novel small molecules against MCF7 cells and identified that compound Z363 led to decreased expression of both MYC and TAF10. The proteasome inhibitor MG132 rescued the expression of both proteins indicating that Z363 promotes their degradation through the proteasome. The authors employed an online tool (UbiBrowser) to predict potential E3 ligases that can target MYC and TAF10. From this, 11 candidate E3 ligases were identified and subsequent knockdown studies demonstrated that TRIP12 could modulate protein levels of both MYC and TAF10. TRIP12 expression increased in response to treatment with Z636 and both Z363 and TRIP12 overexpression similarly inhibited proliferation and promoted apoptosis in MCF7 cells. TRIP12 decreased MYC stability by first modulating MYC phosphorylation at T58 to promote subsequent degradation by TRIP12. Comparatively, oridonin targets MYC through upregulation of the kinase GSK-3 and E3 ligase SCFFBW7 to induce increased T58 phosphorylation and MYC degradation, respectively.3 It will be important for future studies to delineate the intermediates involved in promoting T58 phosphorylation in response to Z363 and investigate whether there is functional redundancy between TRIP12- and SCFFBW7-catalysed ubiquitination of MYC. Z363 was also found to promote TRIP12-mediated degradation of the transcription factor TAF10. This draws similarities with the mode of action of immunomodulatory drugs (IMiDs) which promote CRL4CRBN-mediated ubiquitination of transcription factors IKZF1 and IKZF3 resulting in downregulation of MYC activity and further highlights the therapeutic promise of Z363. Together the findings from this study indicate a model whereby TRIP12 and TAF10 dynamically regulate MYC transcription and protein stability and present Z363 as a potential novel anti-cancer strategy which co-regulates both proteins to suppress MYC. TRIP12 is a HECT domain E3 ligase that is best known for its role in the cell cycle through regulation of the tumour suppressor p14/ARF. It is also implicated in the regulation of cellular processes such as chromatin remodelling, DNA repair and cell differentiation. TRIP12 expression is reported to be overexpressed in a number of cancers, including breast cancer, and has been proposed as a potential therapeutic target.9 Xiong and colleagues introduce TRIP12 as a novel player in regulating both MYC stability and transcriptional activity and identify the small molecule Z363 as an activator of TRIP12 activity. This work encourages further investigation to delineate both the role of TRIP12 in tumourigenesis and the mode of action of Z363. One key avenue for exploration will be to understand the relative contributions of TRIP12 and other E3 ligases in regulating MYC stability and whether they act in a cell type-specific manner. Furthermore, it will be imperative to investigate whether Z363 affects TRIP12-mediated ubiquitination of other known substrates, particularly p14/ARF. Intriguingly, TRIP12 was recently identified as a key promoter of PROTAC-mediated degradation of the BET protein BRD4. TRIP12 functions co-operatively with CRL complexes to generate K29/K48-branched ubiquitin chains, accelerating the degradation of BRD4 which in turn leads to suppression of MYC activity.10 It would be fascinating to explore whether PROTAC-based therapies exhibit enhanced efficacy in tumours with high expression of TRIP12 or indeed whether activation of TRIP12 by Z363 can increase the efficacy of PROTACs. In summary, targeting post-translational regulation of MYC is a promising anti-cancer strategy, however, given the myriad of different pathways involved in regulating MYC activity and stability, a better understanding of this dynamic control and crosstalk will be important to progressing therapeutic strategies.

Introduction Despite continuous advances in therapies over the last few decades, Multiple Myeloma (MM) remains an incurable malignancy with almost all patients developing relapsed/refractory disease. This emphasizes the need for novel approaches that can improve the efficacy of current standard of care treatments. Previous work from our lab and others has identified dysregulation of the E3 ligase HUWE1 in MM and highlighted it's potential as a therapeutic target. Through its interaction with a diverse set of substrates, HUWE1 is implicated in many key cellular processes including stress responses and DNA replication and repair pathways and is being increasingly recognised as a major regulator of MYC activity. In this study, we uncover a novel role for HUWE1 in DNA replication and repair and assess the combination of HUWE1 and proteasome inhibition as a potential therapeutic strategy in MM. Methods MM cell lines were transfected with SMARTvector Inducible Human HUWE1 shRNA or a non-targeting control (NTC) shRNA (Dharmacon, USA). Replicative stress was induced by treatment with 2 mM Hydroxyurea (HU) and assessed using immunofluorescence staining and iPOND (isolation of proteins on nascent DNA) analysis. Viability was assessed using CellTiter® Glo and combination indices (CI) calculated using CompuSyn software. Commercially available HUWE1 inhibitor BI8622 (MedChemExpress) and novel HUWE1 inhibitors synthesised in house were used throughout the study. Results HUWE1 knockdown and/or inhibition in MM cell lines led to an accumulation of cells in S phase, consistent with previous studies indicating that HUWE1 is required for effective DNA replication. Using proteomic profiling and co-immunoprecipitation, we identified and validated Replication Protein A, 70 kDa (RPA70) as a novel substrate of HUWE1. RPA70 is a subunit of the heterotrimeric RPA complex which plays a critical role in the recruitment of DNA repair proteins. RPA70 is the first subunit to translocate to sites of DNA damage where it binds to DNA and recruits the other subunits (RPA32 and RPA14) forming the complex, thus allowing for initiation of DNA repair. We observed significantly less K63-linked ubiquitination of RPA70 (p=0.00213) in the absence of HUWE1, with an associated decrease in both phosphorylation (p=0.0064) and localisation of RPA subunit RPA32 to DNA following treatment with hydroxyurea (HU) to induce replicative stress. Subsequent iPOND analysis determined that this is associated with reduced recruitment of repair proteins, including RAD51, to DNA. Moreover, induced replicative stress in HUWE1 depleted/inhibited cells resulted in significantly higher levels of DNA damage at 6hrs (p=0.00421) and 24hrs (p=0.0219) post HU treatment. A recent study demonstrated that HUWE1 ubiquitination of MYC promotes the formation of MYC multimers that protect stalled replication forks to limit the formation of double strand breaks (DSB) (Nature 2022;612:148-155). In line with this we also observe decreased ubiquitination of MYC in the HUWE1 knockdown cell line compared to the NTC following HU treatment. Finally, we assessed the efficacy of combining HUWE1 inhibitors with bortezomib which is known to impair DSB repair. Dual inhibition of HUWE1 and proteasome activity resulted in synergistic effects (CI values < 1) and a corresponding increase in DNA damage compared to HUWE1 inhibitor or bortezomib alone as measured by levels of ƴH2AX. Conclusion We have identified RPA as a novel substrate for HUWE1 and demonstrate that targeting HUWE1 results in increased replication stress and a dampened DNA repair capacity, underpinned by reduced recruitment of repair proteins. This work outlines a clear role for HUWE1 in genome stability in MM cells and highlights that HUWE1 inhibitors represent a novel anti-myeloma strategy that acts in synergy with bortezomib to exacerbate DNA damage.

Background Multiple Myeloma (MM) is an incurable B cell neoplasm characterised by heightened levels of genomic instability that contribute to both development and progression of the disease. HUWE1, an E3 ubiquitin ligase, has been implicated in the DNA damage response (DDR) and genome integrity. Past studies identified more than 5% of patients present with a HUWE1 mutation. Our lab has determined that both MM patients and cell lines with HUWE1 mutations exhibit increased levels of genomic instability manifested by heightened mutation rates (p=0.0023) and increased incidents of micronuclei formation (p<0.0001). This study aimed to elucidate HUWE1’s role in DNA replication and to determine how it influences DNA repair in MM. Methods Cells were transfected with SMARTvector Inducible Human HUWE1 shRNA or with a non-targeting control (NTC) shRNA (Dharmacon, Chicago IL, USA). Co-immunoprecipitation was carried out using the Co-IP kit (Thermo Fisher). Replicative stress was induced with 2mM Hydroxyurea (HU) and assessed using immunofluorescence. DNA repair was investigated in (2Gy) irradiated HUWE1 knockdown cells by immunofluorescence staining. Results In line with previous studies, we found that knockdown of HUWE1 in MM cell lines led to an S-phase arrest, suggesting a role for HUWE1 in DNA replication. Using proteomic profiling and co-immunoprecipitation we identified novel putative substrates of HUWE1 that are involved in DNA replication and repair. To address HUWE1’s role in the replicative stress response we treated cells with 2mM HU to elicit replication fork stalling and used replication protein A (RPA) foci counts as a measure of the response. We found that HUWE1 depleted cells exhibited significantly less foci and therefore reduced recruitment of replication proteins when treated with HU for 6hrs compared to the NTC control (p=0.0064). This reduced response to replicative stress in HUWE1 knockdown cells was coupled with significantly higher levels of DNA damage at 6hrs (p=0.00421) and this damage persisted at 24hrs after treatment (p=0.0219). To further examine HUWE1’s role in the DDR cells were stained for the double strand break (DSB) marker, 53BP1 following irradiation (IR). HUWE1 knockdown cells displayed a reduced capacity to repair DSBs with more 53BP1 foci present at 1hrs (p=0.00254), 4hrs (p=0.0469) and 24hrs (p=0.025) post-IR compared to their NTC counterparts. Conclusion Here we demonstrate that knockdown of HUWE1 results in increased replication stress and a dampened DNA repair capacity in MM cells, most likely underpinned by reduced recruitment of repair machinery. This data coupled with our previous work demonstrating a role for HUWE1 mutations as a driver for genomic instability, outlines a clear position for HUWE1 in maintaining genome integrity in MM. Further exploration of these dysregulated repair pathways in the presence of HUWE1 mutations may offer potential therapeutic targets for a subset of patients in the future. Multiple Myeloma (MM) is an incurable B cell neoplasm characterised by heightened levels of genomic instability that contribute to both development and progression of the disease. HUWE1, an E3 ubiquitin ligase, has been implicated in the DNA damage response (DDR) and genome integrity. Past studies identified more than 5% of patients present with a HUWE1 mutation. Our lab has determined that both MM patients and cell lines with HUWE1 mutations exhibit increased levels of genomic instability manifested by heightened mutation rates (p=0.0023) and increased incidents of micronuclei formation (p<0.0001). This study aimed to elucidate HUWE1’s role in DNA replication and to determine how it influences DNA repair in MM. Cells were transfected with SMARTvector Inducible Human HUWE1 shRNA or with a non-targeting control (NTC) shRNA (Dharmacon, Chicago IL, USA). Co-immunoprecipitation was carried out using the Co-IP kit (Thermo Fisher). Replicative stress was induced with 2mM Hydroxyurea (HU) and assessed using immunofluorescence. DNA repair was investigated in (2Gy) irradiated HUWE1 knockdown cells by immunofluorescence staining. In line with previous studies, we found that knockdown of HUWE1 in MM cell lines led to an S-phase arrest, suggesting a role for HUWE1 in DNA replication. Using proteomic profiling and co-immunoprecipitation we identified novel putative substrates of HUWE1 that are involved in DNA replication and repair. To address HUWE1’s role in the replicative stress response we treated cells with 2mM HU to elicit replication fork stalling and used replication protein A (RPA) foci counts as a measure of the response. We found that HUWE1 depleted cells exhibited significantly less foci and therefore reduced recruitment of replication proteins when treated with HU for 6hrs compared to the NTC control (p=0.0064). This reduced response to replicative stress in HUWE1 knockdown cells was coupled with significantly higher levels of DNA damage at 6hrs (p=0.00421) and this damage persisted at 24hrs after treatment (p=0.0219). To further examine HUWE1’s role in the DDR cells were stained for the double strand break (DSB) marker, 53BP1 following irradiation (IR). HUWE1 knockdown cells displayed a reduced capacity to repair DSBs with more 53BP1 foci present at 1hrs (p=0.00254), 4hrs (p=0.0469) and 24hrs (p=0.025) post-IR compared to their NTC counterparts. Here we demonstrate that knockdown of HUWE1 results in increased replication stress and a dampened DNA repair capacity in MM cells, most likely underpinned by reduced recruitment of repair machinery. This data coupled with our previous work demonstrating a role for HUWE1 mutations as a driver for genomic instability, outlines a clear position for HUWE1 in maintaining genome integrity in MM. Further exploration of these dysregulated repair pathways in the presence of HUWE1 mutations may offer potential therapeutic targets for a subset of patients in the future.

Abstract Introduction Chromosomal instability is a hallmark of Multiple Myeloma (MM), with most patients displaying cytogenetic abnormalities which can arise due to DNA damage response (DDR) defects. TRIM33 is an E3 ligase and transcriptional co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 plays a role in the DDR and can regulate chromosomal stability, but its precise function remains unknown. In this study we investigated the impact of TRIM33 loss in MM on genomic stability and DDR pathways and whether this could be exploited therapeutically. Methods The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with overall survival (OS) and structural variants. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells. The effect on DDR signalling was determined by western blotting and immunofluorescence. The Selleckchem DNA Damage/Repair Compound Library was screened on the JJN3 model in a high-throughput manner using the CellTox™ Green cytotoxicity assay. Validation of selected compounds was performed using CellTiter® Glo viability assay or clonogenic assays. Combination indices (CI) were calculated using CompuSyn software. Results Data on CN, OS and structural variants were available for 730 newly diagnosed MM patients and of these, 69 (9.5%) were identified to have a CN loss of TRIM33. These patients have poorer OS compared to those without TRIM33 loss (52.3 months vs 72.6 months; p<0.0001). Moreover, they exhibit a significantly higher median number of structural variants (deletions, duplications, inversions, and translocations; 38 vs 26; p<0.0001), indicative of increased chromosomal instability. Our data in MM cell lines has shown that TRIM33 is rapidly recruited to chromatin within 5 minutes of induced DNA damage. TRIM33 knockdown led to an increase in 53BP1 foci formation and endogenous γH2AX (P<0.001) indicating unrepaired DNA double-strand breaks (DSBs) typical of a DDR defect. In response to these DSBs both ATM and ATR kinases were activated as demonstrated by increased pKAP1 Ser824 and pCHK1 Ser345 respectively (p<0.001). Additionally, we observed a reduction in RAD51 (p<0.05) indicative of a potential defect in the DSB repair pathway homologous recombination (HR). To identify therapeutic vulnerabilities relating to TRIM33 loss, we performed a high-throughput screen to assess sensitivity to 160 unique DNA damaging compounds. TRIM33 knockdown cells exhibited increased sensitivity to 27 compounds across a range of drug classes. Additional studies confirmed that compared to control cells, TRIM33 knockdown sensitized cells to the PARP inhibitor Olaparib and ATR inhibitors BAY-1895344 and VE-821. Further investigation with VE-821 demonstrated that whilst treatment induced PARP cleavage and DSBs in both control and knockdown cells within 48 hours, knockdown cells exhibited significantly more pCHK1 Ser345 inhibition (p<0.01). Furthermore, combining VE-821 with bortezomib yielded synergistic effects in TRIM33 knockdown cells across a range of doses (CI range 0.57-0.9) while no synergy was observed in control cells (CI>1 for all combinations). Conclusion We have identified a subset of MM patients with TRIM33 loss who display high-risk disease characterized by chromosomal abnormalities and defective DDR. Alongside this we have identified PARP and ATR inhibitors as therapeutic vulnerabilities in cell line models of TRIM33 loss. Moreover, we demonstrate that ATR inhibition increases the efficacy of bortezomib in TRIM33 knockdown cells. Further investigation into these compounds could lead to novel therapies for patients with TRIM33 loss. Disclosures No relevant conflicts of interest to declare.

Multiple Myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells. Deletion of chromosome 1p (del1p) is a common genetic event associated with high-risk MM. Located within this region at 1p13.2 is TRIM33, a chromatin-associated E3 ligase which can function as a transcriptional co-repressor. Recent studies have shown that TRIM33 functions in the PARP-dependent DNA Damage Response (DDR). However, its molecular function during the DDR remains unclear. Here, we investigated the impact of TRIM33 loss on genome stability and the DDR in MM. Using the publicly available CoMMpass (Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile) dataset, we identified 69 (9.5%) out of 730 newly diagnosed MM patients that had a copy number loss of TRIM33. Kaplan-Meier analysis revealed that these patients have a poorer overall survival compared to those without TRIM33 loss (median 52.3 months vs 72.6 months; p<0.0001). In addition, these patients have a significantly higher number of structural variants (median 38 vs 26; p<0.0001) indicative of increased chromosomal instability. We show that TRIM33 is rapidly recruited to chromatin following 2Gy irradiation, where it transiently interacts with the chromatin remodelling enzyme ALC1. Western blotting and immunofluorescence staining revealed an increase in double-strand break markers γH2AX and 53BP1 (p<0.001) in TRIM33 knockdown cells, along with increased expression of key HR protein RAD51 (p<0.01). MM patients with loss of TRIM33 similarly exhibited significantly higher gene expression of 53BP1 and RAD51 (p<0.0001) indicating a potential HR dysregulation. We have demonstrated that TRIM33 loss results in chromosomal instability and increased endogenous DNA damage in MM, typical of a DDR defect. Further understanding the role of TRIM33 in the DDR may lead to opportunities to therapeutically exploit DDR defects in patients with TRIM33 loss.

Background Chromosomal instability is a hallmark of Multiple Myeloma (MM) with most patients displaying cytogenetic abnormalities which can often act as prognostic indicators. Such abnormalities can arise due to defects in the DNA Damage Response (DDR). TRIM33 is an E3 ligase and transcription co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 is involved in PARP-dependent DDR and regulation of chromosomal stability. Here, we investigated the influence of TRIM33 loss in MM, focusing on its role in the DDR and whether this could be exploited therapeutically. Methods The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with survival, structural variants and common cytogenetic abnormalities. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells for in vitro studies. Protein expression and interactions were assessed by co-immunoprecipitation, western blotting and/or immunofluorescence. Clonogenic survival assays were used to assess response to Olaparib. Results Previously we identified a subset of MM patients with TRIM33 loss and identified that these patients exhibit significantly more chromosomal structural variants (deletions, inversions, duplications and translocations) (p<0.0001) and a significantly poorer overall survival (p<0.0001). Additionally, we have determined the frequency of common recurrent primary and secondary cytogenetic abnormalities for these patients. No recurrent primary cytogenetic abnormalities were associated with TRIM33 loss. However, high-risk secondary chromosome 1 aberrations were associated with loss of TRIM33, both del(1p) and gain(1q) (p<0.0001 and p=0.0474 respectively). In vitro, TRIM33 knockdown resulted in increased formation of 53BP1 foci and increased γH2AX expression (p<0.001) indicating unrepaired DNA damage typical of a DDR defect. Following induced DNA damage using 2Gy irradiation (IR), TRIM33 is recruited to chromatin within 5 minutes, with levels returning to basal by 30 minutes. The chromatin remodelling enzyme ALC1 is known to regulate sensitivity to PARP inhibition and TRIM33 is required for its timely removal from sites of damage. TRIM33 transiently interacts with ALC1 within 15 minutes of 2Gy IR. TRIM33 knockdown did not affect ALC1 expression. However, knockdown did sensitize MM cells to the PARP inhibitor Olaparib reducing the IC50 from 1.7µM to 780nM. Conclusion Here, we show that a subgroup of MM patients have TRIM33 loss and these patients have high-risk disease and poor outcome. We show that TRIM33 is recruited to damaged chromatin where it regulates ALC1 activity. Therefore, TRIM33 loss results in a DDR defect leading to chromosomal abnormalities. However, TRIM33 loss-associated DDR defects can be exploited therapeutically using Olaparib which is currently approved for the treatment of BRCA1/2 mutated breast and ovarian cancers. Chromosomal instability is a hallmark of Multiple Myeloma (MM) with most patients displaying cytogenetic abnormalities which can often act as prognostic indicators. Such abnormalities can arise due to defects in the DNA Damage Response (DDR). TRIM33 is an E3 ligase and transcription co-repressor located on chromosome 1p13.2, a region frequently deleted in MM. Previous studies have shown that TRIM33 is involved in PARP-dependent DDR and regulation of chromosomal stability. Here, we investigated the influence of TRIM33 loss in MM, focusing on its role in the DDR and whether this could be exploited therapeutically. The CoMMpass dataset (IA15 release) was screened to identify patients with copy number (CN) loss of TRIM33 and this was correlated with survival, structural variants and common cytogenetic abnormalities. TRIM33 shRNA knockdown models were established in JJN3 and U266 cells for in vitro studies. Protein expression and interactions were assessed by co-immunoprecipitation, western blotting and/or immunofluorescence. Clonogenic survival assays were used to assess response to Olaparib. Previously we identified a subset of MM patients with TRIM33 loss and identified that these patients exhibit significantly more chromosomal structural variants (deletions, inversions, duplications and translocations) (p<0.0001) and a significantly poorer overall survival (p<0.0001). Additionally, we have determined the frequency of common recurrent primary and secondary cytogenetic abnormalities for these patients. No recurrent primary cytogenetic abnormalities were associated with TRIM33 loss. However, high-risk secondary chromosome 1 aberrations were associated with loss of TRIM33, both del(1p) and gain(1q) (p<0.0001 and p=0.0474 respectively). In vitro, TRIM33 knockdown resulted in increased formation of 53BP1 foci and increased γH2AX expression (p<0.001) indicating unrepaired DNA damage typical of a DDR defect. Following induced DNA damage using 2Gy irradiation (IR), TRIM33 is recruited to chromatin within 5 minutes, with levels returning to basal by 30 minutes. The chromatin remodelling enzyme ALC1 is known to regulate sensitivity to PARP inhibition and TRIM33 is required for its timely removal from sites of damage. TRIM33 transiently interacts with ALC1 within 15 minutes of 2Gy IR. TRIM33 knockdown did not affect ALC1 expression. However, knockdown did sensitize MM cells to the PARP inhibitor Olaparib reducing the IC50 from 1.7µM to 780nM. Here, we show that a subgroup of MM patients have TRIM33 loss and these patients have high-risk disease and poor outcome. We show that TRIM33 is recruited to damaged chromatin where it regulates ALC1 activity. Therefore, TRIM33 loss results in a DDR defect leading to chromosomal abnormalities. However, TRIM33 loss-associated DDR defects can be exploited therapeutically using Olaparib which is currently approved for the treatment of BRCA1/2 mutated breast and ovarian cancers.

Faithful DNA replication during cellular division is essential to maintain genome stability and cells have developed a sophisticated network of regulatory systems to ensure its integrity. Disruption of these control mechanisms can lead to loss of genomic stability, a key hallmark of cancer. Ubiquitination is one of the most abundant regulatory post-translational modifications and plays a pivotal role in controlling replication progression, repair of DNA and genome stability. Dysregulation of the ubiquitin proteasome system (UPS) can contribute to the initiation and progression of neoplastic transformation. In this review we provide an overview of the UPS and summarize its involvement in replication and replicative stress, along with DNA damage repair. Finally, we discuss how the UPS presents as an emerging source for novel therapeutic interventions aimed at targeting genomic instability, which could be utilized in the treatment and management of cancer.

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

Genomic instability is a hallmark of cancer cells which results in excessive DNA damage. To counteract this, cells have evolved a tightly regulated DNA damage response (DDR) to rapidly sense DNA damage and promote its repair whilst halting cell cycle progression. The DDR functions predominantly within the context of chromatin and requires the action of chromatin-binding proteins to coordinate the appropriate response. TRIM24, TRIM28, TRIM33 and TRIM66 make up the transcriptional intermediary factor 1 (TIF1) family of chromatin-binding proteins, a subfamily of the large tripartite motif (TRIM) family of E3 ligases. All four TIF1 proteins are aberrantly expressed across numerous cancer types, and increasing evidence suggests that TIF1 family members can function to maintain genome stability by mediating chromatin-based responses to DNA damage. This review provides an overview of the TIF1 family in cancer, focusing on their roles in DNA repair, chromatin regulation and cell cycle regulation.

Genomic instability is a hallmark of Multiple Myeloma (MM), with almost all patients displaying cytogenetic abnormalities including ploidy changes, deletions, amplifications and translocations. A common recurrent genetic event in MM with prognostic significance is the deletion of chromosome 1p. TRIM33, an E3 ligase, is located within this deleted region at 1p13. Recent studies demonstrate a role for TRIM33 in the PARP-dependent DNA Damage Response (DDR) and showed that loss of TRIM33 results in the accumulation of chromosomal abnormalities. TRIM33 functions as a tumour suppressor in a number of cancer types, including chronic myelomonocytic leukaemia and hepatocellular carcinoma. Low TRIM33 expression has previously been associated with poor overall survival in MM (GSE2658), however, little is known about its molecular function in MM.

Genomic instability is a prominent feature in the development and progression of Multiple Myeloma (MM). The E3 ligase HUWE1 contributes to the regulation of genomic stability by promoting DNA damage tolerance during replicative stress. Previous studies, by our lab and others, have shown dysregulation of HUWE1 in MM. We have demonstrated an increase in expression of HUWE1 across plasma cell dyscrasias and HUWE1 has been reported as a mutational driver in t(11;14) myeloma. The aim of this study was to investigate the role of HUWE1 in DNA replication and explore whether dysregulation of HUWE1 contributes to genomic instability in MM.