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The oncogenic transcription factor c-Maf has been proposed as an ideal therapeutic target for multiple myeloma (MM), but how to achieve it is still elusive. In the present study, we found the Otub1/c-Maf axis could be a potential target. Otub1, an OTU family deubiquitinase, was found to interact with c-Maf by mass spectrometry. Otub1 abrogates c-Maf K48-linked polyubiquitination, thus preventing its degradation and enhancing its transcriptional activity. Specifically, this deubiquitinating activity depends on its Lys71 and the N terminus but is independent of UBE2O, a known E2 of c-Maf. Otub1 promotes MM cell survival and MM tumor growth. In contrast, silence of Otub1 leads to c-Maf degradation and c-Maf-expressing MM cell apoptosis. Therefore, the Otub1/c-Maf axis could be a therapeutic target of MM. In order to explore this concept, we performed a c-Maf recognition element–driven luciferase-based screen against US Food and Drug Administration–approved drugs and natural products, from which the generic cardiac glycoside lanatoside C (LanC) is found to prevent c-Maf deubiquitination and induces its degradation by disrupting the interaction of Otub1 and c-Maf. Consequently, LanC inhibits c-Maf transcriptional activity, induces c-Maf-expressing MM cell apoptosis, and suppresses MM growth and prolongs overall survival of model mice, but without apparent toxicity. Therefore, the present study identifies Otub1 as a novel deubiquitinase of c-Maf and establishes that the Otub1/c-Maf axis is a potential therapeutic target for MM.

Introductions Although intratumor heterogeneity and clonal evolution have been inferred in multiple myeloma (MM), this was largely focused at the bulk tumor population level. Single-cell analysis is of significant importance in delineating the exact phylogeny of subclonal population and in discovering subtle diversification. Here, we identified the clonal architecture of different time points using multi-gene fluorescence in situ hybridization (mFISH) at single cell level, and explored the prognostic values of different clonal evolution patterns in MM. Methods We performed mFISH in 129 longitudinal samples of 57 MM patients. All the patients had newly-diagnosed and relapsed paired samples, and 12 patients had cytogenetic evaluation for more than two time points. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. Results 43 of 57 patients (75.4%) harbored three or four cytogenetic clones at diagnosis. We delineated the phylogeny of subclonal tumor population in each patient and established robust trends for the timing of temporal acquisition in the whole cohort using the pairwise precedence. 13q deletion and the first 1q gain tended to be earlier cytogenetic alternation, whereas 16q and 17p deletion were acquired later. The sequence of 13q deletion and 1q21 gain occurrence was identified in 23 patients by the single-cell analysis. 1q21 gain and 13q deletion each occurred first in 12 and 11 patients respectively. Strikingly, patients in whom 13q deletion was acquired first showed a significantly worse survival than 1q21 gain-first patients (median OS 32.9 vs. 71.2 months, p=0.010). We inferred the most likely ancestral relationships between subclones and derived the evolutionary architecture in each patient. Four distinct evolutionary patterns were identified (Figure 1). 18 of 57 (31.6%) patients showed clonal stabilization. These patients were characterized by no novel subclones emerging and no existed subclones disappearing at relapse. Differential evolution was observed in 12 patients, where clonal dynamics resulted from a change in predominant clone from presentation to relapse. The major clone at diagnosis disappeared or decreased to a minor clone while a subclone showed growth advantage and turned to be a major clone at relapse. We found evidence of branching evolution in 9 patients. Here, one or more clones harboring novel cytogenetic abnormalities emerged between the early and late time points, whereas some disappeared. The remainder of patients demonstrated a linear evolution pattern (18/57, 31.6%). The predominant clones acquired one or more novel cytogenetic abnormalities at the later time point. Patients with clonal stabilization had a significantly improved OS than those with other evolutionary patterns (median OS, 71.2 vs. 39.7 vs. 35.2 vs. 25.5 months, for stable, differential, branching and linear patterns, respectively, p=0.001). However, there is no difference in sampling interval among four evolutionary patterns (p=0.131). Therefore, the survival differences were mostly attributable to a significantly shorter failure free survival from relapse (p In order to evaluate the accuracy of abnormalities detection by mFISH, we performed cFISH in these 57 MM patients. Cell fractions of cytogenetic abnormalities detected by mFISH were significantly correlated with that detected by cFISH (p Conclusions These findings suggest that mFISH is a valuable tool for the analysis of clonal phylogeny and evolution pattern of critical cytogenetic aberrations. Patients may benefit from the repeated cytogenetic evaluation, especially for the risk stratification of survival after relapse. Personalized treatment strategy is required for MM patients based on their clonal evolution patterns. Figure 1 Disclosures No relevant conflicts of interest to declare.

Myeloproliferative neoplasms (MPNs), particularly polycythemia vera (PV) and essential thrombocythemia (ET), are often associated with a high risk of acquired arterial and venous thrombosis. MPNs are also considered as vascular diseases that occur frequently in elderly patients, often accompanied by acute coronary syndrome (ACS).Very few studies have been made to estimate the risk after percutaneous coronary intervention (PCI) in MPNs with ACS patients. Moreover, the operative procedures or perioperative management procedures have not yet been fully established. The purpose of this study is to evaluate the risk factors and the effectiveness of PCI in MPNs with ACS. Methods: From February 2001 through November 2018, a total of 215 patients were admitted to Beijing An Zhen Hospital with objectively proven ET (n=137) or PV (n=78). Among the 51 MPNs patients with ACS, 46 had undergone PCI therapy. Preoperative anticoagulation and perioperative antiplatelet therapy in all patients were similar to those in cardiovascular patients following the guideline. Patients with PCI were divided into two groups, the emergency PCI (24h). Clinical data including medical history, age, gender, whole blood counts, JAK2v617F mutation, the type of ACS, vascular risk factors and target vessels disease and complications of PCI were retrospectively studied and the risk factors were analyzed using multivariate logistic regression analysis. Results: Among the 46 patients with PCI, 37 were proven ET and 9 PV, including 27 cases of acute myocardial infarction (AMI) and 19 cases of unstable angina pectoris (UA). Their mean age is 58.22±12.10. Leukocyte and platelet count were higher than normal (WBC11.22±5.23╳109/L, HCT43.75±7.13%, PLT599.02±280.37╳109/L). The positive rate of JAK2V617F mutation was 61.11% (22/36), with 4 patients with CALR mutation. According to thrombosis risk stratification, 32.61% and 67.39% of the patients were classified as low risk and high risk thrombophilias respectively. 82.61% (38/46) of the patients had cardiovascular risk factor, while 52.61% showed two risk factors or above. 15 patients (32.61%) had a history of thrombosis. Nearly half of the patients (22 cases, or 47.83%) were diagnosed simultaneously with MPN because of ACS. Coronary angiography showed 69 branch lesions, and left anterior descending artery(LAD)was the most common crime vessel (55.07%,38/69), followed by the right coronary artery (RCA) (24.64%, or 17/69). 29 cases (63.04%) were single vessel lesions. 11 (23.91%) cases were accompanied by coronary artery thrombus formation. Patients received a total of 61 stents (60 drug-eluting stents and 1 bare metal stents) implantation. There were 10 cases of complications in the perioperative period (21.74%), including 4 cases of postoperative stent thrombosis, 1 with ventricular fibrillation, 2 of re-infarction , 2 of arterial dissection, 1 of hemorrhagic local hematoma and 1 with low blood volume. Most complications of these cases occurred within 4 days after the operation, but no patients died. Among patients treated with PCI therapy, 27 and 19 of the patients received emergency and elective surgery, respectively. There were no significant differences in the gender, MPNs type, vascular risk factors, thrombosis history, crime vessel, the number of stent implantation and complications between the two groups. The proportion of patients with WBC>10×109/L , single vessel lesions or WBC count was higher in patients with emergency surgery than that in patients with elective surgery. Logistic regression analysis showed that thrombosis history (OR=27.235, P=0.034; 95%CI, 1.286-575.262) and coronary artery thrombus formation (OR=39.359, P=0.012; 95%CI, 2.223-696.778 ) were independent risk factors for complications of PCI in the perioperative period. Conclusion: Our data show that PCI treatment, especially emergency surgery is not uncommon in MPN patients. Emergency PCI should be relatively safe and effective. Although there was a high occurrence of post-PCI complications and stent re-thrombosis, there were no fatal or serious complications. It is suggested that individualized cytoreduction therapy be given first, and once blood cells count decreases, PCI complications may significantly reduce. Previous thrombus history and coronary artery thrombus were independent risk factors for complications of PCI in the perioperative period. Disclosures No relevant conflicts of interest to declare.