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ObjectiveThe objective of this study was to determine the prevalence of benign vocal fold lesions (BVFLs) in patients with chronic cough over a 1‐year period.Study DesignCase series with chart review.SettingTertiary academic medical center.Subjects and MethodsA retrospective cohort study of patients with chronic cough seen in our tertiary multidisciplinary cough clinic from 2016 to 2017 was conducted. Patient characteristics, presence of BVFLs by laryngoscopy, and Leicester Cough Questionnaire (LCQ) were recorded.ResultsA total of 419 patients were included (average age: 61 years), and 67% of patients were female. Ten percent of patients had BVFLs: granuloma (5%), leukoplakia (3%), nodules (2%), and polyps (1%). Median cough duration was 2.9 years (interquartile range [IQR], 0.7‐8.5); no significant difference in median cough duration was observed between patients with BVFLs and those with no lesions (2.6 vs 3.0 years, P =. 86). In total, 178 patients (42%) had available LCQ data: median score of 10.2 (IQR, 7.9‐13.9); no significant difference in median LCQ was observed between patients with BVFLs and those with no lesions (10.5 vs 10.1, P =. 54).ConclusionPrevalence of BVFLs in our cohort of patients with chronic cough was 10%, with posterior glottic lesions—granuloma—being the most common (5%). Prevalence of midmembranous lesions was as follows: vocal fold nodules (2%) and vocal fold polyps (1%). BVFLs were not associated with longer cough duration or more severe cough. We cannot determine a causative or noncausative relationship between BVFLs and chronic cough at this time due to lack of a control group.

Abstract Background Although 20 pancreatic cancer susceptibility loci have been identified through genome-wide association studies in individuals of European ancestry, much of its heritability remains unexplained and the genes responsible largely unknown. Methods To discover novel pancreatic cancer risk loci and possible causal genes, we performed a pancreatic cancer transcriptome-wide association study in Europeans using three approaches: FUSION, MetaXcan, and Summary-MulTiXcan. We integrated genome-wide association studies summary statistics from 9040 pancreatic cancer cases and 12 496 controls, with gene expression prediction models built using transcriptome data from histologically normal pancreatic tissue samples (NCI Laboratory of Translational Genomics [n = 95] and Genotype-Tissue Expression v7 [n = 174] datasets) and data from 48 different tissues (Genotype-Tissue Expression v7, n = 74–421 samples). Results We identified 25 genes whose genetically predicted expression was statistically significantly associated with pancreatic cancer risk (false discovery rate < .05), including 14 candidate genes at 11 novel loci (1p36.12: CELA3B; 9q31.1: SMC2, SMC2-AS1; 10q23.31: RP11-80H5.9; 12q13.13: SMUG1; 14q32.33: BTBD6; 15q23: HEXA; 15q26.1: RCCD1; 17q12: PNMT, CDK12, PGAP3; 17q22: SUPT4H1; 18q11.22: RP11-888D10.3; and 19p13.11: PGPEP1) and 11 at six known risk loci (5p15.33: TERT, CLPTM1L, ZDHHC11B; 7p14.1: INHBA; 9q34.2: ABO; 13q12.2: PDX1; 13q22.1: KLF5; and 16q23.1: WDR59, CFDP1, BCAR1, TMEM170A). The association for 12 of these genes (CELA3B, SMC2, and PNMT at novel risk loci and TERT, CLPTM1L, INHBA, ABO, PDX1, KLF5, WDR59, CFDP1, and BCAR1 at known loci) remained statistically significant after Bonferroni correction. Conclusions By integrating gene expression and genotype data, we identified novel pancreatic cancer risk loci and candidate functional genes that warrant further investigation.

LINKED CONTENTThis article is linked to Wang et al and Rej et al papers. To view these articles, visit https://doi.org/10.1111/apt.15419 and https://doi.org/10.1111/apt.15517.

Background: Mechanical injury in the conduction system requiring permanent pacemaker (PPM) associated with transcatheter aortic valve replacement (TAVR) procedure is a common complication. The objective of this study was to use ambulatory monitor BodyGuardian to assess late occurrence of atrioventricular block (AVB) after TAVR. Methods: This prospective study evaluated 365 patients who underwent TAVR at Mayo Clinic, Rochester, Minnesota between June 2016 and August 2017. Patients who received PPM for bradycardia after TAVR before discharge were considered as the PPM group. Those not requiring PPM received a BodyGuardian system (BodyGuardian group) for 30 days of continuous monitoring. Primary end point was Mobitz II or third-degree atrioventricular block (II/III AVB) at 30-day follow-up. Results: Of 365 patients, 74 who had a PPM or an implantable cardioverter-defibrillator before TAVR and 94 who were enrolled in other studies were excluded. Of 197 patients enrolled in the study, 70 (35.5%) received PPM and 127 had BodyGuardian before the hospital dismissal. Eleven of 127 (8.6%) BodyGuardian group required PPM within 30 days after TAVR for late occurrence of symptomatic bradycardia. In total, 33 of 197 (16.7%) patients developed II/III AVB (24 before and 9 after discharge). Thirty-four patients had preexisting right bundle branch block. Of them, 16 (47%) developed II/III AVB. Of 53 patients who developed new left bundle branch block after TAVR, 14% progressed to II/III AVB within 30 days. Conclusions: In patients without a standard post-TAVR pacing indication, yet a potential risk to develop AVB, a strategy of 30-day monitoring identifies additional patients who require permanent pacing.

Introduction . There is a paucity of information about risk behaviors in adolescents with chronic kidney disease (CKD). We designed this study to assess the prevalence of risk behaviors among teens with CKD in the United States and to investigate any associations between risk behavior and patient or disease characteristics. Methods . After informed consent, adolescents with CKD completed an anonymous, confidential, electronic web-based questionnaire to measure risk behaviors within five domains: sex, teen driving, alcohol and tobacco consumption, illicit drug use, and depression-related risk behavior. The reference group was composed of age-, gender-, and race-matched US high school students. Results . When compared with controls, teens with CKD showed significantly lower prevalence of risk behaviors, except for similar use of alcohol or illicit substances during sex (22.5% vs. 20.8%, p = 0.71 ), feeling depressed for ≥2 weeks (24.3% vs. 29.1%, p = 0.07 ), and suicide attempt resulting in injury needing medical attention (36.4% vs. 32.5%, p = 0.78 ). Furthermore, the CKD group had low risk perception of cigarettes (28%), alcohol (34%), marijuana (50%), and illicit prescription drug (28%). Use of two or more substances was significantly associated with depression and suicidal attempts ( p < 0.05 ) among teens with CKD. Conclusions . Teens with CKD showed significantly lower prevalence of risk behaviors than controls. Certain patient characteristics were associated with increased risk behaviors among the CKD group. These data are somewhat reassuring, but children with CKD still need routine assessment of and counselling about risk behaviors.

OBJECTIVE As compensation transitions from a fee-for-service to pay-for-performance healthcare model, providers must prioritize patient-centered experiences. Here, the authors’ primary aim was to identify predictors of patient dissatisfaction at 1 and 2 years after lumbar surgery. METHODS The Michigan Spine Surgery Improvement Collaborative (MSSIC) was queried for all lumbar operations at the 1- and 2-year follow-ups. Predictors of patients’ postoperative contentment were identified per the North American Spine Surgery (NASS) Patient Satisfaction Index, wherein satisfied patients were assigned a score of 1 (“the treatment met my expectations”) or 2 (“I did not improve as much as I had hoped, but I would undergo the same treatment for the same outcome”) and unsatisfied patients were assigned a score of 3 (“I did not improve as much as I had hoped, and I would not undergo the same treatment for the same outcome”) or 4 (“I am the same or worse than before treatment”). Multivariable Poisson generalized estimating equation models were used to report adjusted risk ratios (RR adj ). RESULTS Among 5390 patients with a 1-year follow-up, 22% reported dissatisfaction postoperatively. Dissatisfaction was predicted by higher body mass index (RR adj =1.07, p < 0.001), African American race compared to white (RR adj = 1.51, p < 0.001), education level less than high school graduation compared to a high school diploma or equivalent (RR adj = 1.25, p = 0.008), smoking (RR adj = 1.34, p < 0.001), daily preoperative opioid use > 6 months (RR adj = 1.22, p < 0.001), depression (RR adj = 1.31, p < 0.001), symptom duration > 1 year (RR adj = 1.32, p < 0.001), previous spine surgery (RR adj = 1.32, p < 0.001), and higher baseline numeric rating scale (NRS)–back pain score (RR adj = 1.04, p = 0.002). Conversely, an education level higher than high school graduation, independent ambulation (RR adj = 0.90, p = 0.039), higher baseline NRS–leg pain score (RR adj = 0.97, p = 0.013), and fusion surgery (RR adj = 0.88, p = 0.014) decreased dissatisfaction. Among 2776 patients with a 2-year follow-up, 22% reported dissatisfaction postoperatively. Dissatisfaction was predicted by a non-white race, current smoking (RR adj = 1.26, p = 0.004), depression (RR adj = 1.34, p < 0.001), symptom duration > 1 year (RR adj = 1.47, p < 0.001), previous spine surgery (RR adj = 1.28, p < 0.001), and higher baseline NRS–back pain score (RR adj = 1.06, p = 0.003). Conversely, at least some college education (RR adj = 0.87, p = 0.035) decreased the risk of dissatisfaction. CONCLUSIONS Both comorbid conditions and socioeconomic circumstances must be considered in counseling patients on postoperative expectations. After race, symptom duration was the strongest predictor of dissatisfaction; thus, patient-centered measures must be prioritized. These findings should serve as a tool for surgeons to identify at-risk populations that may need more attention regarding effective communication and additional preoperative counseling to address potential barriers unique to their situation.

Abstract The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with >90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p<0.0001). Regarding other genomic aberrations, they were either absent (NOTCH1 mutations) or rare (SF3B1 mutations, trisomy 12, del(11q), TP53 aberrations due to either del(17p) and/or TP53 mutations). The sole exception concerned a high frequency (14%) of TP53 aberrations in subset #29 (p<0.05 compared with the other subsets), which is notable for M-CLL cases in general. Time to first treatment (TTFT) could be analyzed in 228 cases. IgG subsets #4 and #16 had significantly (p=0.036) longer TTFT (median TTFT: not yet reached) compared to the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). In conclusion, we have identified distinct clinicobiological profiles for different stereotyped IGHV4-34 M-CLL subsets, highlighting subsets #4 and #16 as particularly indolent, which is important for both medical and social reasons, especially considering that a significant proportion of patients in these subsets are diagnosed at younger ages. Our findings support the notion that BcR IG stereotypy refines prognostication in CLL, superseding the crude immunogenetic distinction based on SHM load only. Additionally, the observed heterogeneity suggests that not all M-CLL are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Disclosures Tausch: Gilead: Other: Travel support. Shanafelt:Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Cephalon: Research Funding. Niemann:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy; Novartis: Other: Travel grant. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:Janssen Pharmaceuticals: Research Funding.

Abstract Background: Thrombotic and hemorrhagic complications are commonly encountered in uncontrolled essential thrombocythemia (ET). Both anagrelide and hydroxyurea (HU) have proven efficacious in cytoreduction as well as reducing these events and remain first line therapy for most high-risk ET patients. Independent of their role in risk-reduction, little is known about how these therapies impact patient symptomatology or quality of life. In this study, we compared the clinical and symptomatic profiles of ET patients receiving HU or anagrelide against patients with no previous experience with these agents. Methods: Data was assessed from a prospectively collected international database of ET patients in which demographics, disease features, and ET symptoms utilizing the myeloproliferative neoplasm symptom assessment form (MPN-SAF; Scherber et al, 2011). The MPN-SAF includes the patient's perceptions of common MPN-related symptoms and overall quality of life (QOL) on a 0 (absent) to 10 (worst imaginable) scale. Total symptom score (TSS) was computed based on symptom items using the published scoring algorithm on a 0 (all reported symptoms absent) to 100 (all reported symptoms worst imaginable) scale. ET risk scores were calculated using the IPSET scoring algorithm (Passamonti 2012). Thrombocytopenia was defined as a platelet count <100 x 10(9)/L, anemia was defined as hemoglobin<10 g/dL and leukopenia was defined as a white blood cell count <4.0 x 10(9)/L. Associations between the MPN-SAF individual symptoms were investigated using chi-square test for categorical data and ANOVA F-test for continuous variables. Results Hydroxyurea vs. HU Naive A total of 402 ET patients with active HU use were compared to 392 ET patients with no history of HU use. Patients using HU were older (63.5 years vs. 52.3 years, p<0.001) and had a greater concentration of both high risk (24.9% vs. 11.4%, p=0.001) and intermediate risk (53.9% vs 38.4%, p<0.001) patients. They also had a higher rate of prior thrombosis (29.8% vs 11.3%, p<0.001) and leukopenia (10.1% vs. 3.0%, p<0.001). No differences were noted between gender, a history of prior hemorrhage, red blood cell transfusion requirements, or the presence of anemia/thrombocytopenia. In comparing symptom profiles, no significant differences were noted between TSS or individual symptoms with the exception of slightly more severe cough in HU patients (1.5 vs. 1.1, p=0.02, Figure 1). Anagrelide vs. Anagrelide Naive A total of 49 ET patients with active anagrelide use were compared to 794 ET patients with no history of anagrelide use. Patients using anagrelide had a longer mean disease duration (8.1 years vs. 5.8 years) and were more anemic (9.1% vs. 1.2%, p<0.001). No differences were noted between age, gender, risk scores, the presence of leukopenia/thrombocytopenia, a history of prior thrombosis or hemorrhage complications or red blood cell transfusion requirements. Additionally, there were no significant differences between TSS or individual symptom items (Figure 1). HU vs. Anagrelide A total of 402 patients currently using HU were compared to 39 patients currently using anagrelide. Overall, HU users were slightly older (63.5 years vs. 55.1 years, p<0.001) with a greater population of patients meeting high risk criteria (24.9% vs. 2.8%, p=0.002) and having a history of prior thrombosis (29.8% vs. 12.8%, p=0.02). Patients receiving anagrelide had a slightly longer disease duration (8.2 years vs. 6.0 years, p=0.0446). In comparing symptom profiles, no differences were noted in TSS or individual symptom items between cohorts. Discussion In this retrospective analysis, it does not appear cytoreduction with either HU and/or anagrelide has a significant impact on ET symptom burden despite reducing vascular events. Importantly, the higher risk scores in HU patients did not translate directly into greater patient symptomatology supporting previous studies demonstrating a poor association between these two items. Prospective trials measuring ET symptom change, in the setting of randomized trials will better quantify impact of cytoreduction on symptom burden as well as quantify impact of newer agents such as interferon or jak inhibition. Figure 1. Symptom Comparisons Between HU, Anagrelide and Other Therapy Figure 1. Symptom Comparisons Between HU, Anagrelide and Other Therapy Disclosures Kiladjian: Incyte Corporation: Consultancy; Novartis: Other: Travel grant; Research Funding paid to institution (Hôpital Saint-Louis et Université Paris Diderot); Novartis: Consultancy. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Harrison:Sanofi: Honoraria, Speakers Bureau; Shire: Speakers Bureau; Gilead: Honoraria; CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cervantes:Sanofi-Aventis: Consultancy; Novartis: Consultancy, Speakers Bureau; CTI-Baxter: Consultancy, Speakers Bureau. Barbui:Novartis: Speakers Bureau. Etienne:ARIAD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Congress Travel/Accomodations, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Roy:Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding. te Boekhorst:Novartis: Consultancy; CTI Biopharma: Consultancy. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Speakers Bureau; Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees. Mesa:Novartis. Research- incyte, Gilead, cti, Genentech, promedior, NS Pharma: Consultancy.

Introduction We have shown that in myeloma (MM) the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide, in a CRBN dependent fashion, inhibit thioredoxin reductase and thus increase intracellular oxidative stress as a consequence of peroxide accumulation (Sebastian et al. 2017). Accordingly, we have also shown that CRBN expression alone does not fully correlate with lenalidomide sensitivity, as the cells ability to decompose H2O2 is important - MM cells with lower H2O2 decomposition capacity are more sensitive to IMiDs. We hypothezised that the specific metabolic pathways used by cells for biomass production would influence IMiD sensitivity since they also influence this cellular oxidative capacity. Cellular proteins are key elements of this biomass, and required for the proliferation of MM cells and production of immunoglobulin proteins. Methods We cultured MM cells under various nutrients conditions to understand its effect on cell proliferation and drug responses. Cell viability was assessed by MTT assay, the Luminescent Assay, and direct cell counting. MM cells glycolytic rate was measured using the Seahorse XF analyzer. Western blots were performed to quantify various protein expression levels in MM cells cultured under different nutrients conditions. CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and a lentivirus system was used to over-express CRBN in MM cells. Results MM cells resistant to lenalidomide, but still expressing functional CRBN, show a higher glycolytic pathway use than sensitive cells. We found that the lenalidomide sensitive cell line MM1.S is highly dependent on extracellular glutamine for cell proliferation. In contrast, JJN3, a lenalidomide resistant cells line with wild type CRBN, preferentially consumes glucose. We found that glutamine depletion from culture media completely abolished lenalidomide sensitivity in MM1.S cell line. Western blot analysis revealed that antibody production by MM1.S was primarily dependent on glutamine availability, and that JJN3 antibody production was dependent upon glucose availability. While glutamine depletion from culture medium completely abolished lenalidomide sensitivity in MM1.S cell line, IKZF1, and IKZF3 degradation was unchanged. We further studied the role of CRBN in glutamine dependent cellular biomass protein production using isogenic cell lines (+/- wtCRBN). We found that wtCRBN expressing cells proliferate more and show higher antibody production in the presence of glutamine, over CRBN negative isogenic cell lines. Lenalidomide treatment further increased glutamine dependent antibody production in wtCRBN expressing cells. One possibile explanation is that activation of glutamine catabolism can facilitate protein demethylation via the supplementation α-ketoglutarate (α-KG). Therefore, we tested whether lenalidomide treatment could induce protein demethylation in sensitive cell lines. Western blot probed with mono-methyl lysine antibody showed lenalidomide induced protein demethylation. We further confirmed that protein demethylation, and lenalidomide sensitivity is a consequence of elevated α-KG by treating cells with cell-permeable 5-octyl- α-ketoglutarate which act as a substrate of the lysine demethylases. 5-Octyl-α-ketoglutarate treatment inhibited cell proliferation preferentially in wtCRBN expressing cells, and also enhanced lenalidomide induced sensitivity. Protein demethylation is associated with protein ubiquitination and proteasomal degradation. We thus hypothesize that lenalidomide induced protein demethylation also likely increases proteasome inhibitors sensitivity in MM. Conclusion MM cells with preferential glutamine consumption are likely to be more sensitive to lenalidomide, and extracellular glutamine depletion can induce lenalidomide resistance. MM cells expressing CRBN, but not dependent on glutamine for protein biomass production are more likely to be resistant to lenalidomide. Our study postulates that CRBN protein requirement in lenalidomide sensitivity cells is restricted to high glutamine dependency, and that it is quite likely CRBN has a role in glutamine metabolism. Moreover, MM cells that consume more glutamine are under higher oxidative stress and exhibit less H2O2 decomposition capacity and therefore increasing sensitivity to IMiDs. References Sebastian , S., et al., Blood 2017 129:991-1007 Disclosures Stewart: Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Celgene: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board.

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.