Citations

Abstract Mutation of Triggering receptor expressed on myeloid cells 2 (TREM2) impairs the response of microglia to amyloid‐β (Aβ) pathology in Alzheimer's disease (AD), although the mechanism governing TREM2‐regulated microglia recruitment to Aβ plaques remains unresolved. Here, we confirm that TREM2 mutation attenuates microglial migration. Then, using Trem2 −/− mice and an R47H variant mouse model for AD generated for this study, we show that TREM2 deficiency or the AD‐associated R47H mutation results in inhibition of FAK and Rac1/Cdc42‐GTPase signaling critical for cell migration. Intriguingly, treatment with CN04, a Rac1/Cdc42‐GTPase activator, partially enhances microglial migration in response to oligomeric Aβ 42 in Trem2 −/− or R47H microglia both in vitro and in vivo. Our study shows that the dysfunction of microglial migration in the AD‐associated TREM2 R47H variant is caused by FAK/Rac1/Cdc42 signaling disruption, and that activation of this signaling ameliorates impaired microglial migration response to Aβ 42 , suggesting a therapeutic target for R47H‐bearing patients with high risk of AD.

Abstract Blood lipids have been associated with the development of a range of cancers, including breast, lung and colorectal cancer. For endometrial cancer, observational studies have reported inconsistent associations between blood lipids and cancer risk. To reduce biases from unmeasured confounding, we performed a bidirectional, two‐sample Mendelian randomization analysis to investigate the relationship between levels of three blood lipids (low‐density lipoprotein [LDL] and high‐density lipoprotein [HDL] cholesterol, and triglycerides) and endometrial cancer risk. Genetic variants associated with each of these blood lipid levels ( P < 5 × 10 −8 ) were identified as instrumental variables, and assessed using genome‐wide association study data from the Endometrial Cancer Association Consortium (12 906 cases and 108 979 controls) and the Global Lipids Genetic Consortium (n = 188 578). Mendelian randomization analyses found genetically raised LDL cholesterol levels to be associated with lower risks of endometrial cancer of all histologies combined, and of endometrioid and non‐endometrioid subtypes. Conversely, higher genetically predicted HDL cholesterol levels were associated with increased risk of non‐endometrioid endometrial cancer. After accounting for the potential confounding role of obesity (as measured by genetic variants associated with body mass index), the association between genetically predicted increased LDL cholesterol levels and lower endometrial cancer risk remained significant, especially for non‐endometrioid endometrial cancer. There was no evidence to support a role for triglycerides in endometrial cancer development. Our study supports a role for LDL and HDL cholesterol in the development of non‐endometrioid endometrial cancer. Further studies are required to understand the mechanisms underlying these findings.

AbstractPancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.

Abstract We generated eight multiple myeloma cell lines resistant to bortezomib; five acquired PSMB5 mutations. In 1,500 patients such mutations were rare clinically. To better understand disruption of proteasomes on multiple myeloma viability and drug sensitivity, we systematically deleted the major proteasome catalytic subunits. Multiple myeloma cells without PSMB5 were viable. Drug-resistant, PSMB5-mutated cell lines were resensitized to bortezomib by PSMB5 deletion, implying PSMB5 mutation is activating in its drug resistance function. In contrast, PSMB6 knockout was lethal to multiple myeloma cell lines. Depleting PSMB6 prevented splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8, and PSMB10; however, PSMB6 engineered without splicing function or catalytic activity, also restored viability, inferring the contribution of PSMB6 to proteasome structure to be more important than functional activity. Supporting this, bortezomib sensitivity was restored in drug-resistant multiple myeloma cell lines by low level expression of mutated PSMB6 lacking splicing function. Loss of PSMB8 and PSMB9 was neither lethal nor restored bortezomib sensitivity. Significant codependency of PSMB5, PSMB6, and PSMB7 expression was observed. We demonstrated elevated levels of PSMB6 and 7, but not 8 and 9, in some, but not all, serial patient samples exposed to proteasome inhibitors. In summary, we show PSMB6 and PSMB7, but not PSMB5, to be essential for multiple myeloma cell survival, this dependency is structural and that upregulation or activating mutation of PSMB5, 6, and 7 confers proteasome inhibitor resistance, while depletion confers sensitivity. Implications: These findings support modulation of PSMB5, PSMB6, or PSMB7 expression as a new therapeutic strategy.

The significance of peristenotic collateral circulation (PCC) development around a stenotic renal artery is unknown. We tested the hypothesis that PCC is linked to loss of kidney function and recovery potential in patients with atherosclerotic renovascular disease (ARVD). Thirty-four patients with ARVD were assigned to medical-therapy with or without revascularization based on clinical indications. The PCC was visualized using multidetector computed tomography and defined relative to segmental arteries in patients with essential hypertension. PCC number before and 3 months after treatment was correlated with various renal parameters. Thirty-four stenotic kidneys from 30 patients were analyzed. PCC number correlated inversely with kidney volume. ARVD–stenotic kidneys with baseline PCC (collateral ARVD [C-ARVD], n=13) associated with elevated 24-hour urine protein and stenotic kidney vein level of tumor necrosis factor-α, lower single-kidney volume and blood flow, and greater hypoxia than in stenotic kidneys with no PCC (no collateral ARVD [NC-ARVD], n=17). Revascularization (but not medical-therapy alone) improved stenotic kidney function and reduced inflammation in both NC-ARVD and C-ARVD. In C-ARVD, revascularization also increased stenotic kidney volume, blood flow, and oxygenation to levels comparable to NC-ARVD, and induced PCC regression. However, revascularization improved systolic blood pressure, plasma renin activity, and filtration fraction only in NC-ARVD. Therefore, patients with C-ARVD have greater kidney dysfunction, atrophy, hypoxia, and inflammation compared with patients with NC-ARVD, suggesting that PCC does not effectively protect the stenotic kidney in ARVD. Renal artery revascularization improved in C-ARVD stenotic kidney function, but not hypertension or renin-angiotensin system activation. These observations may help direct management of patients with ARVD.

5077 Background: Several ICIs are used in first and subsequent lines of therapy for mRCC, either alone or in combination with another ICI or targeted therapy (TT). There are no data on the efficacy and safety of using an ICI in patients who have already received an ICI in a prior line of therapy. Methods: We reviewed patients with mRCC at 8 institutions who received 2 separate lines of ICI therapy (ICI-1, ICI-2), including as a single-agent and/or combination with other agents. The primary outcomes were overall response rate (ORR) and time to progression (TTP) with ICI-1 and ICI-2. Immune-related adverse events (irAEs) were graded using CTCAEv5.0. Results: 65 patients were included. Median age at diagnosis of mRCC was 60 years (range 30-86) and the majority had clear cell RCC (n=56, 86%). Median follow-up was 3.5 years (95% CI 2.9-4.4). Median lines at which ICI-1 and ICI-2 were received were 1 (1-6) and 3 (2-8) respectively. Reasons for discontinuing ICI-1 were disease progression (n=47, 72%), toxicity (n=15, 23%) or other (n=3, 5%). Therapies received at ICI-2 were single-agent ICI (n=26, 40%), or combinations of ICI with another ICI (n=20, 31%), TT (n=11, 17%) or other agent (n=8, 12%). Responses to ICI-1 and ICI-2 are shown in the Table; ORR to ICI-2 was significantly lower than to ICI-1 (23% vs. 36%, p=0.044). Amongst those who responded to ICI-2 (n=14), 7 (50%) received single-agent ICI, and the remainder received ICI in combination with another ICI (n=4, 29%) or TT (n=3, 21%); 7 patients (50%) had previously responded to ICI-1. The ORR to ICI-2 was higher in responders to ICI-1 (32%) compared to those with SD (17%) or PD (15%) to ICI-1. Median TTP (mTTP) at ICI-2 was shorter compared to ICI-1 (5.3 months vs. 8.5 months, Wilcoxon p=0.024). 29 patients (45%) experienced an irAE with ICI-2; 8 (12%) and 3 (5%) had a grade 3 or 4 irAE respectively, with 3 (30%) of these patients having previously had ≥grade 3 irAE to ICI-1. There were no treatment-related deaths. Conclusions: The ORR to ICI-2 was 23%, which is comparable to that seen with ICI after prior TT. Responses were seen even amongst those receiving single-agent ICI at ICI-2 and the likelihood of response to ICI-2 was higher if a patient had previously responded to ICI-1. No increase in toxicity with ICI-2 was apparent. Additional data from prospective studies are needed to determine whether sequential ICI has a role in treatment of mRCC. [Table: see text]

AbstractEffective therapeutic strategies are needed to preserve renal function in patients with atherosclerotic renal artery stenosis (ARAS). Low‐energy shockwave therapy (SW) and adipose tissue‐derived mesenchymal stem/stromal cells (MSCs) both stimulate angiogenesis repair of stenotic kidney injury. This study tested the hypothesis that intrarenal delivery of adipose tissue‐derived MSCs would enhance the capability of SW to preserve stenotic kidney function and structure. Twenty‐two pigs were studied after 16 weeks of ARAS, ARAS treated with a SW regimen (bi‐weekly for 3 weeks) with or without subsequent intrarenal delivery of adipose tissue‐derived MSCs and controls. Four weeks after treatment, single‐kidney renal blood flow (RBF) before and after infusion of acetylcholine, glomerular filtration rate (GFR), and oxygenation were assessed in vivo and the renal microcirculation, fibrosis, and oxidative stress ex vivo. Mean arterial pressure remained higher in ARAS, ARAS + SW, and ARAS + SW + MSC compared with normal. Both SW and SW + MSC similarly elevated the decreased stenotic kidney GFR and RBF observed in ARAS to normal levels. Yet, SW + MSC significantly improved RBF response to acetylcholine in ARAS, and attenuated capillary loss and oxidative stress more than SW alone. Density of larger microvessels was similarly increased by both interventions. Therefore, although significant changes in functional outcomes were not observed in a short period of time, adjunct MSCs enhanced pro‐angiogenic effect of SW to improve renal microvascular outcomes, suggesting this as an effective stratege for long‐term management of renovascular disease.

Abstract We have conducted systematic small interfering RNA (siRNA) lethality screening of the kinome and “druggable genome” in human myeloma cell lines (HMCL) to functionally generate a comprehensive map of critical genes and vulnerabilities in human myeloma tumors. KMS11 human myeloma cells were screened with both an 1800 oligo (639 gene) siRNA library targeting the kinome and with a 13,984 oligo (6,791 gene) library targeting the druggable genome in duplicate using optimized conditions that resulted in &gt;95% transfection efficiency and &lt;5% background cytotoxicity. siRNA were used at low concentration (13nM) to minimise off-target cellular effects. Each gene was initially screened with at least 2 distinct siRNA. Universally lethal and non-silencing siRNA were tested in parallel as controls. Viability was measured at 96 hours by ATP-dependent luminescence and was normalized to the mean and distribution of controls by the B-score method. Approximately 14% of kinome siRNA and 5.8% of druggable genome siRNA caused important reductions in HMCL viability (greater than three standard deviations from control samples treated with non-silencing siRNA), compared with an anticipated rate due to chance of only 0.135%. Short-listed kinome vulnerabilities were validated by repeat targeting in a second lethality screening experiment in KMS11 using a total of four independent siRNA per gene. Forty-two kinases were confirmed as critical for myeloma cell survival. Unsupervised pathway analysis indicates that approximately half are concentrated within established pathways involved in cytokine (VEGFR, FGFR3, IGFR/IL6R) signaling and regulation of cell cycle, apoptosis or metabolism. The role of other survival kinases in myeloma are novel. The selectivity of kinome vulnerabilities identified in KMS11 for myeloma cells versus other tissues was examined by parallel silencing of these targets in KMS11 and JJN3 myeloma cells and in non-myeloma 293 and A549 epithelial cells. The most lethal kinase targets in myeloma that simultaneously show experimental evidence of tumor selectivity include aurora kinase A (AURKA), G-protein receptor coupled kinase 6 (GRK6) and protein kinase N1 (PKN1). By gene expression analyses, PKN1 and GRK6 are both preferentially expressed in plasma cells, compared with an analysis of &gt;50 somatic tissues in which they are absent or only weakly expressed. The extent to which the kinome vulnerabilities identified in KMS11 occur in other HMCL was assessed by tertiary siRNA screening and by lentiviral shRNA techniques. More than 75% of kinases vulnerable in KMS11 cells are recurrently vulnerable in other myeloma tumor lines. Secondary validation studies of all genomic vulnerabilities identified from the much larger siRNA lethality screen of the druggable genome in myeloma cells is ongoing, however, to date 134 genes have been validated as essential myeloma survival factors during primary screening by concordant lethality of 2/2 independent siRNA (compared with an expected rate due to chance of n&lt;1 genes). Significantly, proteosome subunits and specific ubiquitin ligases number amongst the most potently lethal genomic vulnerabilities in KMS11. Overall, this attempt to systematically and functionally document all genetic vulnerabilities in myeloma provides a strong rational impetus for the development of novel therapies that optimally target susceptibilities inherent within myeloma tumors.

Abstract From a high throughput RNAi screen of the human druggable genome targeting the KMS11 cell line, we identified the suppression of the vacuolar H+- ATPase (V-ATPase) family as cytotoxic to myeloma cells. In the screen, two oligos against each gene for the V-ATPase subunits ATP6V1A and ATP6V1B1 resulted in suppression of cell growth (50% and 60% inhibition of cell viability respectively). We further confirmed this result using both lentiviral shRNA knockdown and two small molecule inhibitors specific for V-ATPase. Silencing of ATP6V1A by lentiviral shRNA knock-down in KMS11 and in OPM1 myeloma cell lines caused 75–80% reduction of cell viability at 5 days post infection (measured by MTT assay). Consistent with this result, the V-ATPase specific inhibitors, bafilomycin A1 and REATA 203, both inhibited the growth of a genetically heterogeneous and standardized panel of 14 human myeloma cell lines in vitro with an IC50 ranging from 2.2 – 8.9 nM (mean 5.25 nM) for bafilomycin A1 and 46–1594 nM (mean 542.5 nM)) for REATA 203. We further demonstrated that patient samples (n=10) were sensitive to 20nM bafilomycin A1 which induced a mean of 58% of MM cells to undergo apoptosis (range 10% to 93%) after 24 hours of treatment. Similar to bafilomycin A1, treatment of primary patient-derived MM cells with 500 nM REATA 203 for 72 hours resulted in a mean 69% apoptosis (range 24% to 97%). In contrast, non-myeloma cells (the CD138- fractions of the bone marrow samples) were less sensitive - mean 9% apoptosis (range from 0% to 34%) under the same treatment conditions. Of high interest, however, unlike most drugs we have studied in pre-clinical myeloma models, the cytotoxicity induced by bafilomycin A1 in MM cell lines is abrogated by co-culture with patient bone marrow stromal cells but is not affected by IL-6 or IGF-1 treatment. Dexamethasone- or melphalan-resistant MM cell lines were also highly sensitive to both bafilomycin A1 and REATA 203. In a xenographic JJN3 mouse model, bafilomycin A1 suppresses and delays growth of tumor in a dose-dependent fashion. Gene expression analysis of normal-donor bone marrow plasma cells (n=19), primary tumor samples from MM patients (n=107) and normal somatic tissues demonstrates ubiquitous expression of most subunits of V-ATPase, however, some subunits are preferentially expressed in myeloma cells compared with normal plasma cells, including ATP6V1F (84% vs. 11%), ATP6V1E1 (29% vs. 5%), ATP6V1G2 (17% vs. 0%) and ATP6V0E 2 (36% vs. 16%). In conclusion, our data indicate that vacuolar H+-ATPase inhibitors are of interest as potential therapeutics for MM.