Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Evolution and host-specific adaptation of Pseudomonas aeruginosa.

The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

Focal Adhesion Kinase and Colony Stimulating Factors: Intestinal Homeostasis and Innate Immunity Crosstalk.

Thousands struggle with acute and chronic intestinal injury due to various causes. Epithelial intestinal healing is dependent on phenotypic transitions to a mobile phenotype. Focal adhesion kinase (FAK) is a ubiquitous protein that is essential for cell mobility. This phenotype change is mediated by FAK activation and proves to be a promising target for pharmaceutical intervention. While FAK is crucial for intestinal healing, new evidence connects FAK with innate immunity and the importance it plays in macrophage/monocyte chemotaxis, as well as other intracellular signaling cascades. These cascades play a part in macrophage/monocyte polarization, maturation, and inflammation that is associated with intestinal injury. Colony stimulating factors (CSFs) such as macrophage colony stimulating factor (M-CSF/CSF-1) and granulocyte macrophage colony stimulating factor (GM-CSF/CSF-2) play a critical role in maintaining homeostasis within intestinal mucosa by crosstalk capabilities between macrophages and epithelial cells. The communication between these cells is imperative in orchestrating healing upon injury. Diving deeper into these connections may allow us a greater insight into the role that our immune system plays in healing, as well as a better comprehension of inflammatory diseases of the gut.

Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.

Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF)ßTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/ßTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated.

Diving Into the Health Problems of Competitive Divers: A Systematic Review of Injuries and Illnesses in Pre-elite and Elite Diving Athletes.

CONTEXT: The Olympic sport of diving involves the competitive disciplines of 3 m springboard and 10 m platform. Although it is generally accepted that lumbar spine injuries are common in diving athletes, the existing literature of health problems in diving athletes remains scarce. OBJECTIVE: To identify the incidence, prevalence, and type of health problems that occur in competitive diving athletes. DATA SOURCES: Medline, EMBASE, SportsDiscus, PsycINFO, and Google Scholar. STUDY SELECTION: Studies written in English investigating elite or pre-elite competitive diving (springboard, platform) injuries and/or illnesses were eligible. Two independent reviewers screened for inclusion by title, abstract, and full text in accordance with the eligibility criteria. STUDY DESIGN: Systematic review. LEVEL OF EVIDENCE: Level 4. DATA EXTRACTION: Data extraction was completed by 1 author using a structured form. A second author then independently reviewed and verified the extracted data, any discrepancies were resolved through consensus. RESULTS: The search identified 2554 potential articles, with 28 studies meeting eligibility criteria. The surveillance setting of most studies was restricted to competition-based events, with the reported injury incidence proportion ranging from 2.1% to 22.2%. The reported injury incidence rate ranged from 1.9 to 15.5 per 1000 athlete-exposures. Injuries to the shoulder, lower back/lumbar spine, trunk, and wrist/hand were reported most frequently. The prevalence of low back pain was reported as high as 89% (lifetime), 43.1% (period), and 37.3% (point). The illness incidence proportion ranged from 0.0% to 22.2%, with respiratory and gastrointestinal illness reported most frequently. CONCLUSION: Up to 1 in 5 diving athletes sustain an injury and/or illness during periods of competition. A reporting bias was observed, with most cohort studies limiting surveillance to short competition-based periods only. This limits the current understanding of the health problems experienced by diving athletes to competition periods only and requires expansion to whole-of-year surveillance.

Mechanisms of USP18 deISGylation revealed by comparative analysis with its human paralog USP41.

The ubiquitin-like protein ISG15 (interferon-stimulated gene 15) regulates the host response to bacterial and viral infections through its conjugation to proteins (ISGylation) following interferon production. ISGylation is antagonized by the highly specific cysteine protease USP18, which is the major deISGylating enzyme. However, mechanisms underlying USP18's extraordinary specificity towards ISG15 remains elusive. Here, we show that USP18 interacts with its paralog USP41, whose catalytic domain shares 97% identity with USP18. However, USP41 does not act as a deISGylase, which led us to perform a comparative analysis to decipher the basis for this difference, revealing molecular determinants of USP18's specificity towards ISG15. We found that USP18 C-terminus, as well as a conserved Leucine at position 198, are essential for its enzymatic activity and likely act as functional surfaces based on AlphaFold predictions. Finally, we propose that USP41 antagonizes conjugation of the understudied ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) from substrates in a catalytic-independent manner. Altogether, our results offer new insights into USP18's specificity towards ISG15, while identifying USP41 as a negative regulator of FAT10 conjugation.

Risk Factors for Trochanteric Bursitis Following Total Hip Arthroplasty: A Radiographic Analysis.

BACKGROUND: Trochanteric bursitis (TB) is a prevalent complication following total hip arthroplasty (THA), with increased offset hypothesized as a potential risk factor. This study investigated potential TB predictors in THA patients, including radiographic measurements of offset and leg length, comorbidities, and patient characteristics. METHODS: In this retrospective cohort study, all THA patients from a single academic tertiary care center between 2005 and 2021 were reviewed. Exclusion criteria included less than one-year follow-up, osteonecrosis, or fracture. Manual radiographic measurements of offset (acetabular, femoral, and total) and leg length from preoperative and postoperative antero-posterior pelvis X-rays were taken, with scaling using femoral cortical diameter. Univariable and multivariable Cox proportional hazard models were employed to estimate TB risk. RESULTS: Of 1,094 patients, 103 (9.4%) developed TB, with a median (Q1, Q3) time to presentation of 41.8 weeks (25.5, 66.9). In univariable models, only sex was associated with increased TB risk, with women exhibiting a 1.79 times increased risk (hazard ratio: 1.79 (1.16, 2.76), P = .009). Changes in acetabular offset, femoral offset, total offset, and leg length between preoperative and postoperative radiographs were not associated with an increased risk of developing TB in the univariate or multivariate models. Furthermore, various offset thresholds were evaluated, with no amount of increased offset showing increased TB risk. CONCLUSIONS: This study found no relationship between femoral, acetabular, or total offset and TB following THA. These findings suggest that surgeons may consider adding offset for increased prosthetic stability in high-risk cases. However, given that this is a retrospective study, the authors are not advocating for the routine use of increased offset. The study identified women as a risk factor with a 1.79 times higher TB risk, highlighting the importance of counseling women patients on this heightened risk.

Endoscopic Drainage of Pancreatic Fluid Collections.

Pancreatic fluid collections (PFCs) are commonly encountered complications of acute and chronic pancreatitis. With the advancement of endoscopic ultrasound (EUS) techniques and devices, EUS-directed transmural drainage of symptomatic or infected PFCs has become the standard of care. Traditionally, plastic stents have been used for drainage, although lumen-apposing metal stents (LAMSs) are now favored by most endoscopists due to ease of use and reduced procedure time. While safety has been repeatedly demonstrated, follow-up care for these patients is critical as delayed adverse events of indwelling drains are known to occur.

Morbidity and Mortality Following Total Hip and Knee Arthroplasty With Spinal Versus General Anesthesia: A Retrospective Analysis.

BACKGROUND: The optimal anesthetic mode in total joint arthroplasty (TJA) has yet to be clearly identified. Patients undergoing TJA may recieve spinal anesthesia (SA) or general anesthesia (GA). While arthroplasty literature indicates differences in postoperative morbidity, hip fracture literature does not show clear superiority of SA or GA. The purpose of this study was to further investigate this relationship and determine if there is a significant difference in morbidity and mortality between GA and SA in patients undergoing primary total joint arthroplasty. METHODS: Patients undergoing primary THA or TKA from February 2007 to February 2021 were retrospectively reviewed, creating four cohorts: THA/GA (n = 1,266), THA/SA (n = 1,084), TKA/GA (n = 882), and THA/SA (n = 2,067). Readmission within 90 days, mortality within 365 days, and thromboembolic events within 30 days postoperatively were compared using logistic regression, controlling for age, body mass index, and Charlson Comorbidity Index. RESULTS: The odds of experiencing a deep venous thrombosis within 30 days postoperatively were elevated in the analysis of both the THA/GA (odds ratio (OR) = 3.1; 95% confidence interval (CI): 1.5 to 7.0; P = .004) and the TKA/GA (OR = 1.9; 95% CI: 1.2 to 3.0; P = .005) groups. Similarly, the risk of pulmonary embolism as higher in the THA/GA cohort (OR = 3.9; 95% CI: 1.2 to 17.3; P = .04). There were also higher odds of mortality within 365 days postoperatively in THA/GA patients (OR = 4.3; 95% CI: 1.7 to 13.0; P = .004). No other differences existed among TKA patients. CONCLUSIONS: Based upon these data, both SA and GA are reasonable options for primary TKA with similar risk profiles. However, GA may be associated with higher rates of deep venous thrombosis in TJA and pulmonary embolism in THA. General anesthesia (GA) was also loosely associated with increased mortality within 1 year of THA, but this result should be considered with caution.

Incidence and Risk Factors for Acid-fast Bacillus/Fungal Culture Positivity in Primary, Conversion, and Revision Hip and Knee Arthroplasty.

INTRODUCTION: Previous literature has reported minimal incidences of positive fungal/AFB cultures, questioning the routine use of these tests. With growing concern for excessive use, predictive factors for patients at higher risk for intraoperative AFB/fungal infections would help surgeons limit unnecessary testing. This study evaluates the positivity rate and predictive factors of positive fungal and/or acid-fast bacillus (AFB) cultures after primary, conversion, or revision hip and knee arthroplasty. METHOD: Two hundred thirty-eight knee and hip procedures were done between January 2007 and 2022 where intraoperative AFB/fungal cultures were obtained. Procedures included primary total knee arthroplasty, primary total hip arthroplasty, conversion, first of two-stage, second of two-stage, irrigation and débridement polyexchange, and aseptic revision. Positivity rates of intraoperative AFB/fungal cultures were calculated as binomial exact proportions with 95% confidence intervals and are displayed as percentages. Univariable generalized linear mixed models estimated the unadjusted effects of demographics, individual comorbid conditions, and procedural characteristics on the logit of positive AFB/fungal cultures. RESULTS: Two hundred thirty-eight knee and hip procedures recorded an overall positivity rate of 5.8% for intraoperative AFB/fungal cultures. Aseptic revisions showed the lowest rates of positivity at 3.6%, while conversions showed the highest rates of positivity at 14.3%. The positivity rates are highest among patients who are male (9.0%), of Hispanic origin (12.0%), with body mass index <30 (6.4%), and a Charlson Comorbidity Index <5 (6.1%). History of a prior infection in the same surgical joint showed statistically significant influence of odds of culture positivity with an odds ratio of 3.47 ( P -value: 0.039). Other demographic factors that we investigated including age, sex, race, ethnicity, body mass index, and Charlson Comorbidity Index did not show any notable influence on AFB/fungal positivity rates. CONCLUSION: These results suggest utility in obtaining routine intraoperative AFB/fungal cultures, given the relatively high positivity and poor predictive factors.

Accuracy of intraoperative approximation of pelvic tilt using preoperative standing radiographs.

Background: Anterior approach surgeons who utilize intraoperative fluoroscopy often try to match a preoperative radiograph as a reference for intraoperative cup position. Every degree of inaccuracy in tilt leads to a roughly 0.7° change in anteversion. This study aimed to determine how closely pelvic tilt (PT) is approximated intraoperatively when compared to preoperative anteroposterior (AP) radiographs. Methods: This was a retrospective review of 193 primary THA's done by 2 surgeons at an academic tertiary referral center between September 2021-January 2023. There were 24 patients excluded for distorted anatomy, post-traumatic arthritis, insufficient x-rays, or a sacroiliac joint that could not be visualized on film. Data collected included age and BMI. PT was calculated using the formula, Tilt = -(ln((B/A) x (1/0.483)))/0.051. Value A is the distance from the base of the SI joint to the superior margin of the obturator foramen; value B is the height of the obturator foramen. Results: Mean preoperative PT was 0.2° versus intraoperative PT was 3.4° (p < 0.001). Mean absolute difference was 6.5°. 48% of patients (n = 81) had an absolute difference less than 5°, 31% (n = 52) between 5° and 10°, 14% (n = 24) between 10° and 15°, and 7% (n = 12) greater than 15°. There was no correlation between BMI or age and PT discrepancy. Conclusion: Of the patients, 21% had a discrepancy of 10° or greater between their preoperative radiographs and intraoperative fluoroscopic images. Surgeons should be aware of potential errors in cup positioning and be particularly diligent in high-risk cases.

Inpatient Hospital Costs, Emergency Department Visits, and Readmissions for Revision Hip and Knee Arthroplasty.

BACKGROUND: Revision total hip arthroplasty (THA) and total knee arthroplasty (TKA) tremendously burden hospital resources. This study evaluated factors influencing perioperative costs, including emergency department (ED) visits, readmissions, and total costs-of-care within 90 days following revision surgery. METHODS: A retrospective analysis of 772 revision TKAs and THAs performed on 630 subjects at a single center between January 2007 and December 2019 was conducted. Cost data were available from January 2015 to December 2019 for 277 patients. Factors examined included comorbidities, demographic information, preoperative Anesthesia Society of Anesthesiologists score, implant selection, and operative indication using mixed-effects linear regression models. RESULTS: Among 772 revisions (425 THAs and 347 TKAs), 213 patients required an ED visit, and 90 required hospital readmission within 90 days. There were 22.6% of patients who underwent a second procedure after their initial revision. Liver disease was a significant predictor of ED readmission for THA patients (multivariable odds ratio [OR]: 3.473, P = .001), while aseptic loosening, osteolysis, or instability significantly reduced the odds of readmission for TKA patients (OR: 0.368, P = .014). In terms of ED visits, liver disease increased the odds for THA patients (OR: 1.845, P = .100), and aseptic loosening, osteolysis, or instability decreased the odds for TKA patients (OR: 0.223, P < .001). Increased age was associated with increased costs in both THA and TKA patients, with significant cost factors including congestive heart failure for TKA patients (OR: $7,308.17, P = .004) and kidney disease for THA patients. Revision surgeries took longer than primary ones, with TKA averaging 3.0 hours (1.6 times longer) and THA 2.8 hours (1.5 times longer). CONCLUSIONS: Liver disease increases ED readmission risk in revision THA, while aseptic loosening, osteolysis, or instability decreases it in revision TKA. Increased age and congestive heart failure are associated with increased costs. These findings inform postoperative care and resource allocation in revision arthroplasty. LEVEL OF EVIDENCE: Economic and Decision Analysis, Level IV.

Evaluating the impact of genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) on hospital infection prevention and control decisions.

Genomic epidemiology enhances the ability to detect and refute methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in healthcare settings, but its routine introduction requires further evidence of benefits for patients and resource utilization. We performed a 12 month prospective study at Cambridge University Hospitals NHS Foundation Trust in the UK to capture its impact on hospital infection prevention and control (IPC) decisions. MRSA-positive samples were identified via the hospital microbiology laboratory between November 2018 and November 2019. We included samples from in-patients, clinic out-patients, people reviewed in the Emergency Department and healthcare workers screened by Occupational Health. We sequenced the first MRSA isolate from 823 consecutive individuals, defined their pairwise genetic relatedness, and sought epidemiological links in the hospital and community. Genomic analysis of 823 MRSA isolates identified 72 genetic clusters of two or more isolates containing 339/823 (41 %) of the cases. Epidemiological links were identified between two or more cases for 190 (23 %) individuals in 34/72 clusters. Weekly genomic epidemiology updates were shared with the IPC team, culminating in 49 face-to-face meetings and 21 written communications. Seventeen clusters were identified that were consistent with hospital MRSA transmission, discussion of which led to additional IPC actions in 14 of these. Two outbreaks were also identified where transmission had occurred in the community prior to hospital presentation; these were escalated to relevant IPC teams. We identified 38 instances where two or more in-patients shared a ward location on overlapping dates but carried unrelated MRSA isolates (pseudo-outbreaks); research data led to de-escalation of investigations in six of these. Our findings provide further support for the routine use of genomic epidemiology to enhance and target IPC resources.

A survey of experts to identify methods to detect problematic studies: Stage 1 of the INSPECT-SR Project.

Background: Randomised controlled trials (RCTs) inform healthcare decisions. Unfortunately, some published RCTs contain false data, and some appear to have been entirely fabricated. Systematic reviews are performed to identify and synthesise all RCTs which have been conducted on a given topic. This means that any of these 'problematic studies' are likely to be included, but there are no agreed methods for identifying them. The INSPECT-SR project is developing a tool to identify problematic RCTs in systematic reviews of healthcare-related interventions. The tool will guide the user through a series of 'checks' to determine a study's authenticity. The first objective in the development process is to assemble a comprehensive list of checks to consider for inclusion. Methods: We assembled an initial list of checks for assessing the authenticity of research studies, with no restriction to RCTs, and categorised these into five domains: Inspecting results in the paper; Inspecting the research team; Inspecting conduct, governance, and transparency; Inspecting text and publication details; Inspecting the individual participant data. We implemented this list as an online survey, and invited people with expertise and experience of assessing potentially problematic studies to participate through professional networks and online forums. Participants were invited to provide feedback on the checks on the list, and were asked to describe any additional checks they knew of, which were not featured in the list. Results: Extensive feedback on an initial list of 102 checks was provided by 71 participants based in 16 countries across five continents. Fourteen new checks were proposed across the five domains, and suggestions were made to reword checks on the initial list. An updated list of checks was constructed, comprising 116 checks. Many participants expressed a lack of familiarity with statistical checks, and emphasized the importance of feasibility of the tool. Conclusions: A comprehensive list of trustworthiness checks has been produced. The checks will be evaluated to determine which should be included in the INSPECT-SR tool.

A rare case of taper junction corrosion in semi-constrained total knee arthroplasty.

Metallosis is a known yet rare late complication of unicompartmental and total knee arthroplasty (TKA), usually secondary to either metal-backed patellar component failure, mobile-bearing polyethylene dislocation, or catastrophic polyethylene failure and wear through. The majority of literature surrounding metallosis has been published in relation to total hip arthroplasty (THA) metal on metal bearing wear or mechanically assisted crevice corrosion.This case report describes the development of metallosis in a 77-year-old male patient with advanced (Kellgren-Lawrence Grade 4) osteoarthritis with associated valgus deformity, who underwent index TKA with a semiconstrained revision knee system due to intraoperative medial collateral ligament laxity. The taper junction between the titanium alloy stem and cobalt chromium femoral component was the source of diffuse intra-articular metallosis.

Integrin-αvß6 targeted peptide-toxin therapy in a novel αvß6-expressing immunocompetent model of pancreatic cancer.

Previously we reported that a novel αvß6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvß6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvß6. We report that orthotopic implantation of the αvß6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

Protocol for the development of a tool (INSPECT-SR) to identify problematic randomised controlled trials in systematic reviews of health interventions.

INTRODUCTION: Randomised controlled trials (RCTs) inform healthcare decisions. It is now apparent that some published RCTs contain false data and some appear to have been entirely fabricated. Systematic reviews are performed to identify and synthesise all RCTs that have been conducted on a given topic. While it is usual to assess methodological features of the RCTs in the process of undertaking a systematic review, it is not usual to consider whether the RCTs contain false data. Studies containing false data therefore go unnoticed and contribute to systematic review conclusions. The INveStigating ProblEmatic Clinical Trials in Systematic Reviews (INSPECT-SR) project will develop a tool to assess the trustworthiness of RCTs in systematic reviews of healthcare-related interventions. METHODS AND ANALYSIS: The INSPECT-SR tool will be developed using expert consensus in combination with empirical evidence, over five stages: (1) a survey of experts to assemble a comprehensive list of checks for detecting problematic RCTs, (2) an evaluation of the feasibility and impact of applying the checks to systematic reviews, (3) a Delphi survey to determine which of the checks are supported by expert consensus, culminating in, (4) a consensus meeting to select checks to be included in a draft tool and to determine its format and (5) prospective testing of the draft tool in the production of new health systematic reviews, to allow refinement based on user feedback. We anticipate that the INSPECT-SR tool will help researchers to identify problematic studies and will help patients by protecting them from the influence of false data on their healthcare. ETHICS AND DISSEMINATION: The University of Manchester ethics decision tool was used, and this returned the result that ethical approval was not required for this project (30 September 2022), which incorporates secondary research and surveys of professionals about subjects relating to their expertise. Informed consent will be obtained from all survey participants. All results will be published as open-access articles. The final tool will be made freely available.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.